Defensin polynucleotides and methods of use

ABSTRACT

Methods and compositions for modulating development and defense responses are provided. Nucleotide sequences encoding defensin proteins are provided. The sequences can be used in expression cassettes for modulating development, developmental pathways, and defense responses. Transformed plants, plant cells, tissues, and seed are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/123,896, filed May 6, 2005, which is a divisional of U.S. patentapplication Ser. No. 10/178,213, filed Jun. 21, 2002, now U.S. Pat. No.6,911,577, which claims the benefit of U.S. Provisional Application No.60/300,152, filed Jun. 22, 2001 and U.S. Provisional Application No.60/300,241, filed Jun. 22, 2001, all of which are hereby incorporated intheir entirety by reference herein.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of 343769SequenceListing.txt, a creation date of May 6, 2005, and asize of 419 KB. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated in its entirety by referenceherein.

FIELD OF INVENTION

The invention relates to the field of the genetic manipulation ofplants, particularly the modulation of gene activity and development inplants and increased disease resistance.

BACKGROUND OF THE INVENTION

Disease in plants is caused by biotic and abiotic causes. Biotic causesinclude fungi, viruses, bacteria, and nematodes. An example of theimportance of plant disease is illustrated by phytopathogenic fungi,which cause significant annual crop yield losses as well as devastatingepidemics. Plant disease outbreaks have resulted in catastrophic cropfailures that have triggered famines and caused major social change. Allof the approximately 300,000 species of flowering plants are attacked bypathogenic fungi; however, a single plant species can be host to only afew fungal species, and similarly, most fungi usually have a limitedhost range. Generally, the best strategy for plant disease control is touse resistant cultivars selected or developed by plant breeders for thispurpose. However, the potential for serious crop disease epidemicspersists today, as evidenced by outbreaks of the Victoria blight of oatsand southern corn leaf blight. Molecular methods of crop protection havethe potential to implement novel mechanisms for disease resistance andcan also be implemented more quickly than traditional breeding methods.Accordingly, molecular methods are needed to supplement traditionalbreeding methods to protect plants from pathogen attack.

A host of cellular processes enable plants to defend themselves againstdiseases caused by pathogenic agents. These defense mechanisms areactivated by initial pathogen infection in a process known aselicitation. In elicitation, the host plant recognizes apathogen-derived compound known as an elicitor; the plant then activatesdisease gene expression to limit further spread of the invadingorganism. It is generally believed that to overcome these plant defensemechanisms, plant pathogens must find a way to suppress elicitation aswell as to overcome more physically-based barriers to infection, such asreinforcement and/or rearrangement of the actin filament networks nearthe cell's plasma membrane.

Thus, the present invention solves needs for enhancement of the plant'sdefensive elicitation response via a molecularly based mechanism whichcan be quickly incorporated into commercial crops.

SUMMARY OF THE INVENTION

Compositions and methods relating to disease resistance are provided.Particularly, isolated nucleic acid molecules having nucleotide andamino acid sequences for defensins from plants are provided. Thenucleotide sequences of the invention encode small cysteine-richproteins and are variously annotated or described as defensins,defensin-like proteins, antimicrobial peptides, anti-pathogenicpeptides, thionins, antifungal peptides, protease inhibitors, amylaseinhibitors, scorpion toxin-like proteins and small cysteine-richpeptides. They are referred to herein as defensins as they exhibitsimilarity in primary structure to insect defensins. Transformed plantscan be obtained having altered metabolic states with respect to thedefense response.

The defensin genes of the present invention may find use in enhancingthe plant pathogen defense system. The compositions and methods of theinvention can be used for enhancing resistance to plant pathogensincluding fungal pathogens, plant viruses, microorganisms, nematodes,insects, and the like. The method involves stably transforming a plantwith a nucleotide sequence capable of modulating the plant pathogendefense system operably linked with a promoter capable of drivingexpression of a gene in a plant cell. The defensin genes additionallyfind use in manipulating these processes in transformed plants and plantcells.

Transformed plants, plant cells, and seeds, as well as methods formaking such plants, plant cells, and seeds are additionally provided. Itis recognized that a variety of promoters will be useful in theinvention, the choice of which will depend in part upon the desiredlevel of expression of the disclosed genes. It is recognized that thelevels of expression can be controlled to modulate the levels ofexpression in the plant cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an alignment of several defensin genes and a consensusdefensin sequence (SEQ ID NO:469). Zm-PDF1 is set forth in SEQ IDNO:465; Zm-PDF2 is set forth in SEQ ID NO:463; Zm-PDF3 is set forth inSEQ ID NO:235; Zm-PDF4 is set forth in SEQ ID NO:1; Zm-PDF6 is set forthin SEQ ID NO:4; and Zm-PDF13 is set forth in SEQ ID NO:10.

DETAILED DESCRIPTION OF THE INVENTION Overview

The present invention provides, inter alia, compositions and methods formodulating the total level of polypeptides of the present inventionand/or altering their ratios in a plant. By “modulation” an increase ordecrease in a particular character, quality, substance, or response isintended.

The compositions comprise nucleotide and amino acid sequences fromnumerous plant species. Particularly, the nucleotide and amino acidsequences for 85 groups of defensins are provided. By “plant defensingenes” is intended genes that are structurally related to plantdefensins, and include thionins, small cysteine-rich peptides,proteinase inhibitors, amylase inhibitors, and the like. They are calleddefensin genes after a structural classification of proteins (SCOP)classification system. Defensins play a role in defense, morespecifically plant defense against pathogens, and they share similarityin primary and secondary structure with insect defensins. Defensins ofthe invention are classified in the superfamily of Scorpion toxin-likeproteins and in the Plant Defensin family. While not bound by anymechanism of action, expression of the sequences and related genesaround disease induced lesions may control symptom development, as in ahypersensitive response (HR), by controlling the protease mediated celldeath mechanism. The compositions may also function directly asantipathogenic proteins by inhibiting proteases produced by pathogens orby binding cell wall components of pathogens. Thirdly, they may also actas amphipathic proteins that perturb membrane function, leading tocellular toxicity of the pathogens. The defensins are generally smallcysteine-rich peptides and demonstrate antimicrobial activity. By“antimicrobial” or “antimicrobial activity” antibacterial, antiviral,nematocidal, insecticidal, or and antifungal activity is intended.Accordingly, the polypeptides of the invention may enhance resistance toinsects and nematodes. Any one defensin exhibits a spectrum ofantimicrobial activity that may involve one or more antibacterial,antifungal, antiviral, insecticidal, nematocidal, or antipathogenicactivities. They may also be useful in regulating seed storage proteinturnover and metabolism.

Plant defensins generally comprise about 45-54 amino acids with fourdisulfide bridges (Broekaert et al. (1995) Plant Physiol. (Bethesda)108:1353-1358). The defensins of the invention inhibit the growth of abroad range of pathogens, including but not limited to fungi,nematocides, bacteria, insects, and viruses at micromolarconcentrations. Thus, by “defensin-like activity” it is intended thatthe peptides inhibit pathogen growth or damage caused by a variety ofpathogens, including but not limited to, fungi, insects, nematodes,viruses and bacteria. Defensins inhibit pathogen damage through avariety of mechanisms including, but not limited to, alteration ofmembrane ion permeability and induction of hyphal branching in fungaltargets (Garcia-Olmeda et al. (1998) Biopolymers, Peptide Science47:479-491, herein incorporated by reference).

The compositions of the invention can be used in a variety of methodswhereby the protein products can be expressed in crop plants to functionas antimicrobial proteins. Expression will result in alterations ormodulation of the level, tissue, or timing of expression to achieveenhanced disease, insect, nematode, viral, fungal, or stress resistance.The compositions of the invention may be expressed in the native speciesincluding, but not limited to Arachis hypogaea, Vitis vinifera, Licaniamichauxii, Cyamopsis tetragonoloba, Parthenium argentatum, Nicotianabenthamiana, Eucalyptus grandis, Tropaeolum majus, Ricinus communis,Vernonia mespilifolia, Chrysobalanus icaco, Glycine max, Triticumaestivum, Oryza sativa, Zea mays, Brassica napus, Tulipa gesneriana,Beta vulgaris, Allium porrum, Amaranthus retroflexus, Hedera helix,Picramnia pentandra, Taraxacum kok-saghyz., Tulipa fosteriana, Momordicacharantia, or alternatively, can be heterologously expressed in anyplant of interest. In this manner, the coding sequence for the defensincan be used in combination with a promoter that is introduced into acrop plant. In one embodiment, a high-level expressing constitutivepromoter may be utilized and would result in high levels of expressionof the defensin. In other embodiments, the coding sequence may beoperably linked to a tissue-preferred promoter to direct the expressionto a plant tissue known to be susceptible to a pathogen. Likewise,manipulation of the timing of expression may be utilized. For example,by judicious choice of promoter, expression can be enhanced early inplant growth to prime the plant to be responsive to pathogen attack.Likewise, pathogen inducible promoters can be used wherein expression ofthe defensin is turned on in the presence of the pathogen.

If desired, a transit peptide can be utilized to direct cellularlocalization of the protein product. In this manner, the native transitpeptide or a heterologous transit peptide can be used. However, it isrecognized that both extracellular expression and intracellularexpression are encompassed by the methods of the invention.

Sequences of the invention, as discussed in more detail below, encompasscoding sequences, antisense sequences, and fragments and variantsthereof. Expression of the sequences of the invention can be used tomodulate or regulate the expression of corresponding defensin proteins.

The defensin genes of the present invention additionally find use inenhancing the plant pathogen defense system. The compositions andmethods of the invention can be used for enhancing resistance to plantpathogens including fungal pathogens, plant viruses, insect pathogens,bacterial pathogens, nematodes, and the like. The method involves stablytransforming a plant with a nucleotide sequence capable of modulatingthe plant pathogen defense system operably linked with a promotercapable of driving expression of a gene in a plant cell. By “enhancingresistance” increasing the tolerance of the plant to pathogens isintended. That is, the defensin may slow or prevent pathogen infectionand/or spread.

Compositions

Compositions of the invention include nucleotide sequences that havebeen identified as defensins. Defensins are involved in defense responseand development. In particular, the present invention provides forisolated nucleic acid molecules comprising nucleotide sequences encodingthe amino acid sequences shown in SEQ ID NO:2, 3, 5, 6, 8, 9, 11, 12,14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39,41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66,68, 69, 71, 72, 74, 75, 77, 78, 80, 81, 83, 84, 86, 87, 89, 90, 92, 93,95, 96, 98, 99, 101, 102, 104, 105, 107, 108, 110, 111, 113, 114, 116,117, 119, 120, 122, 123, 125, 126, 128, 129, 131, 132, 134, 135, 137,138, 140, 141, 143, 144, 146, 147, 149, 150, 152, 153, 155, 156, 158,159, 161, 162, 164, 165, 167, 168, 170, 171, 173, 174, 176, 177, 179,180, 182, 183, 185, 186, 188, 189, 191, 192, 194, 195, 197, 198, 200,201, 203, 204, 206, 207, 209, 210, 212, 213, 215, 216, 218, 219, 221,222, 224, 225, 227, 228, 230, 231, 233, 234, 236, 237, 239, 240, 242,243, 245, 246, 248, 249, 251, 252, 254, 255, 257, 258, 260, 261, 263,264, 266, 267, 269, 270, 272, 273, 275, 276, 278, 279, 281, 282, 284,285, 287, 288, 290, 291, 293, 294, 296, 297, 299, 300, 302, 303, 305,306, 308, 309, 311, 312, 314, 315, 317, 318, 320, 321, 323, 324, 326,327, 329, 330, 332, 333, 335, 336, 338, 339, 341, 342, 344, 345, 347,348, 350, 351, 353, 354, 356, 357, 359, 360, 362, 363, 365, 366, 368,369, 371, 372, 374, 375, 377, 378, 380, 381, 383, 384, 386, 387, 389,390, 392, 393, 395, 396, 398, 399, 401, 402, 404, 405, 407, 408, 410,411, 413, 414, 416, 417, 419, 420, 422, 423, 425, 426, 428, 429, 431,432, 434, 435, 437, 438, 440, 441, 443, 444, 446, 447, 449, 450, 452,453, 455, 456, 458, 459, 461, 462, 464, 466, or 468. In particular theinvention provides the mature polypeptides having the amino acidsequences set forth in SEQ ID NO:3, 6, 9, 12, 15, 18, 21, 24, 27, 30,33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84,87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129,132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171,174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213,216, 219, 222, 225, 228, 231, 234, 237, 240, 243, 246, 249, 252, 255,258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297,300, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339,342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381,384, 387, 390, 393, 396, 399, 402, 405, 408, 411, 414, 417, 420, 423,426, 429, 432, 435, 438, 441, 444, 447, 450, 453, 456, 459, or 462.Further provided are polypeptides having an amino acid sequence encodedby a nucleic acid molecule described herein, for example those set forthin SEQ ID NO:1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43,46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97,100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139,142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181,184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223,226, 229, 232, 235, 238, 241, 244, 247, 250, 253, 256, 259, 262, 265,268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 307,310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, 343, 346, 349,352, 355, 358, 361, 364, 367, 370, 373, 376, 379, 382, 385, 388, 391,394, 397, 400, 403, 406, 409, 412, 415, 418, 421, 424, 427, 430, 433,436, 439, 442, 445, 448, 451, 454, 457, 460, 463, 465, or 467.

The nucleotide sequences of the invention are sequences comprising aprotein superfamily including defensins, thionins, protease inhibitors,amylase inhibitors, scorpion toxin-like proteins, and smallcysteine-rich peptides. The claimed sequences are members of the plantdefensin class of genes and polypeptides. The plant defensins areidentified herein as “CS” followed by a three-digit number. Thedefensins of the invention fall into 85 groups based on sequencehomology. As indicated elsewhere herein, some of the maize plantdefensins are identified as “Zm-PDF” for Zea mays plant defensins anddesignated as numbers ZmPDF or PDF (e.g. Zm-PDF1 or PDF1). The Zm-PDFand PDF nomenclature has been described previously in U.S. PatentApplications Nos. 60/300,152, and 60/300,241, both filed Jun. 22, 2001,and herein incorporated by reference in their entirety.

The defensins of the invention are aligned to a diverse set of mostlyplant, some non-plant and some animal, proteinase-inhibitors, thionins,especially gamma-thionins, and defensins, and antifungal proteins. Aconsensus sequence (SEQ ID NO:469) for some of the maize nucleotidesequences of the invention can be seen in FIG. 1. There are homologs tothese sequences in soybeans, rice, wheat, and other crops. Theyrepresent a diverse and conserved supergene family in plants.

Group 1 comprises three nucleotide sequences, of which CS004 (SEQ IDNO:1, PDF4) is the representative sequence. The CS004 polypeptide (SEQID NOS:2 and 3), encoded by the nucleotide sequence set forth in SEQ IDNO:1, is expressed in a tassel-preferred pattern. The full-length CS004polypeptide (SEQ ID NO:2) appears to include a signal peptide, whichwhen processed yields a mature polypeptide having the amino acidsequence set forth in SEQ ID NO:3. CS004 is extracellular. CS006 (SEQ IDNO:4, PDF6) appears to be preferentially expressed in the tassel. Thefull length CS006 polypeptide (SEQ ID NO:5) appears to include a signalpeptide, which when processed yields a mature polypeptide having theamino acid sequence set forth in SEQ ID NO:6. CS006 is extracellular.The CS031 nucleotide sequence (SEQ ID NO:7) was obtained from Oryzasativa. The full length CS031 polypeptide (SEQ ID NO:8) appears toinclude a signal peptide, which when processed yields a maturepolypeptide having the amino acid sequence set forth in SEQ ID NO:9.

Group 2 comprises CS013 (SEQ ID NO:10, PDF13), a maize gene. PDF13 iswidely expressed. The full length CS013 polypeptide (SEQ ID NO:11) isexpected to have a signal peptide, thus the mature protein (SEQ IDNO:12) would be extracellular.

Group 3 comprises two maize sequences, of which CS017 (SEQ ID NO:13,PDF17) is the representative sequence. The CS017 sequence appears to bepreferentially expressed in kernels. The full length CS017 (SEQ IDNO:14) appears to include a signal peptide, which when processed yieldsa mature polypeptide having the amino acid sequence set forth in SEQ IDNO:15. Thus, the predicted CS017 polypeptide would be extracellular.CS018 (SEQ ID NO:16, PDF18) is expressed in the kernel endosperm and maybe expressed elsewhere. The full length polypeptide (SEQ ID NO:17)appears to include a signal peptide, which when processed yields amature polypeptide having the amino acid sequence set forth in SEQ IDNO:18.

Group 4 comprises 13 sequences of which CS065 (SEQ ID NO:43) is therepresentative sequence. CS065, having the nucleotide sequence set forthin SEQ ID NO:43 and encoding the polypeptides set forth in SEQ ID NOS:44and 45, was isolated from Triticum aestivum. The nucleotide sequencesfor CS040 (SEQ ID NO:25), CS041 (SEQ ID NO:28), CS051 (SEQ ID NO:31),CS057 (SEQ ID NO:34), CS059 (SEQ ID NO:37), CS062 (SEQ ID NO:40), CS066(SEQ ID NO:46), CS069 (SEQ ID NO:49), CS070 (SEQ ID NO:52), and CS073(SEQ ID NO:55) were isolated from Triticum aestivum also. Thesenucleotide sequences encode full length polypeptides having the aminoacid sequences set forth in SEQ ID NOS:26, 29, 32, 35, 38, 41, 47, 50,53, and 56, which when processed yield mature polypeptides having theamino acid sequences set forth in SEQ ID NOS:27, 30, 33, 36, 39, 42, 48,51, 54, and 57, respectively. The nucleotide sequences for CS028 (SEQ IDNO:19) and CS029 (SEQ ID NO:22) were isolated from Oryzae sativa. Thesenucleotide sequences encode full length polypeptides having the aminoacid sequences set forth in SEQ ID NOS:20 and 23, which when processedyield mature polypeptides having the amino acid sequences set forth inSEQ ID NOS:21 and 24, respectively.

Group 5 comprises four sequences of which CS032 (SEQ ID NO:58) is therepresentative sequence. CS032, having the nucleotide sequence set forthin SEQ ID NO:58 and encoding the full-length and mature polypeptides setforth in SEQ ID NOS:59 and 60, was isolated from Glycine max. Thenucleotide sequences for CS034 (SEQ ID NO:61) and CS035 (SEQ ID NO:64)were isolated from Glycine max also. These nucleotide sequences encodefull length polypeptides having the amino acid sequences set forth inSEQ ID NOS:62 and 65, which when processed yield mature polypeptideshaving the amino acid sequences set forth in SEQ ID NOS:63 and 66,respectively. The nucleotide sequences for CS052 (SEQ ID NO:67) wasisolated from Triticum aestivum. This nucleotide sequence encodes a fulllength polypeptide having the amino acid sequence set forth in SEQ IDNO:68, which when processed yields a mature polypeptide having the aminoacid sequences set forth in SEQ ID NO:69.

Group 6 comprises two Triticum aestivum sequences of which CS044 (SEQ IDNO:70) is the representative sequence. CS044 (SEQ ID NO:70) and CS050(SEQ ID NO:73) encode full length polypeptides having the amino acidsequences set forth in SEQ ID NOS:71 and 74, which when processed yieldmature polypeptides having the amino acid sequences set forth in SEQ IDNOS:72 and 75, respectively.

Group 7 comprises two Triticum aestivum sequences of which CS074 (SEQ IDNO:79) is the representative sequence. CS063 (SEQ ID NO:76) and CS074(SEQ ID NO:79) encode full length polypeptides having the amino acidsequences set forth in SEQ ID NOS:77 and 80, which when processed yieldmature polypeptides having the amino acid sequences set forth in SEQ IDNOS:78 and 81, respectively.

Group 8 comprises two Beta vulgaris sequences of which CS078 (SEQ IDNO:85) is the representative sequence. CS079 (SEQ ID NO:82) and CS078(SEQ ID NO:85) encode full length polypeptides having the amino acidsequences set forth in SEQ ID NOS:83 and 86, which when processed yieldmature polypeptides having the amino acid sequences set forth in SEQ IDNOS:84 and 87, respectively.

Group 9 comprises CS084 (SEQ ID NO:88), a Hedera helix sequence. Thisnucleotide sequence encodes a full length polypeptide having the aminoacid sequence set forth in SEQ ID NO:89, which when processed yields amature polypeptide having the amino acid sequence set forth in SEQ IDNO:90.

Group 10 comprises two sequences of which CS091 (SEQ ID NO:91) is therepresentative sequence. CS091 (SEQ ID NO:91) and CS098 (SEQ ID NO:94)encode full length polypeptides having the amino acid sequences setforth in SEQ ID NOS:92 and 95, which when processed yield maturepolypeptides having the amino acid sequences set forth in SEQ ID NOS:93and 96, respectively. CS091 was isolated from Tulipa fosteriana, andCS098 was isolated from Tulipa gesneriana.

Group 11 comprises four sequences of which CS092 (SEQ ID NO:100) is therepresentative sequence. CS092, having the nucleotide sequence set forthin SEQ ID NO:100 and encoding the full-length and mature polypeptidesset forth in SEQ ID NOS:101 and 102, was isolated from Tulipafosteriana. CS094 (SEQ ID NO:97), CS093 (SEQ ID NO:103), and CS099 (SEQID NO:106) encode full length polypeptides having the amino acidsequences set forth in SEQ ID NOS:98, 104, and 107, which when processedyield mature polypeptides having the amino acid sequences set forth inSEQ ID NOS:99, 105, and 108, respectively.

Group 12 comprises two Tulipa gesneriana sequences of which CS097 (SEQID NO:112) is the representative sequence. CS102 (SEQ ID NO:109) andCS097 (SEQ ID NO:112) encode full length polypeptides having the aminoacid sequences set forth in SEQ ID NOS:110 and 113, which when processedyield mature polypeptides having the amino acid sequences set forth inSEQ ID NOS:111 and 114, respectively.

Group 13 comprises two Tulipa gesneriana sequences of which CS101 (SEQID NO:115) is the representative sequence. CS101 (SEQ ID NO:115) andCS154 (SEQ ID NO:118) encode full length polypeptides having the aminoacid sequences set forth in SEQ ID NOS:116 and 119, which when processedyield mature polypeptides having the amino acid sequences set forth inSEQ ID NOS:117 and 120, respectively.

Group 14 comprises two Momordica charantia sequences of which CS104 (SEQID NO:121) is the representative sequence. CS104 (SEQ ID NO:121) andCS105 (SEQ ID NO:124) encode full length polypeptides having the aminoacid sequences set forth in SEQ ID NOS:122 and 125, which when processedyield mature polypeptides having the amino acid sequences set forth inSEQ ID NOS:123 and 126, respectively.

Group 15 comprises two Nicotiana benthamiana sequences of which CS112(SEQ ID NO:127) is the representative sequence. CS112 (SEQ ID NO:127)and CS166 (SEQ ID NO:130) encode full length polypeptides having theamino acid sequences set forth in SEQ ID NOS:128 and 131, which whenprocessed yield mature polypeptides having the amino acid sequences setforth in SEQ ID NOS:129 and 132, respectively.

Group 16 comprises two sequences of which CS128 (SEQ ID NO:133) is therepresentative sequence. CS128 (SEQ ID NO:133) and CS153 (SEQ ID NO:136)encode full length polypeptides having the amino acid sequences setforth in SEQ ID NOS:134 and 137, which when processed yield maturepolypeptides having the amino acid sequences set forth in SEQ ID NOS:135and 138, respectively. CS128 was isolated from Tulipa fosteriana, andCS153 was isolated from Tulipa gesneriana.

Group 17 comprises 24 Taraxacum kok-saghyz sequences of which CS130 (SEQID NO:142) is the representative sequence. CS129 (SEQ ID NO:139), CS130(SEQ ID NO:142), CS131 (SEQ ID NO:145), CS132 (SEQ ID NO:148), CS133(SEQ ID NO:151), CS134 (SEQ ID NO:154), CS135 (SEQ ID NO:157), CS136(SEQ ID NO:160), CS137 (SEQ ID NO:163), CS138 (SEQ ID NO:166), CS139(SEQ ID NO:169), CS140 (SEQ ID NO:172), CS141 (SEQ ID NO:175), CS142(SEQ ID NO:178), CS143 (SEQ ID NO:181), CS144 (SEQ ID NO:184), CS145(SEQ ID NO:187), CS146 (SEQ ID NO:190), CS147 (SEQ ID NO:193), CS148(SEQ ID NO:196), CS149 (SEQ ID NO:199), CS150 (SEQ ID NO:202), CS151(SEQ ID NO:205), and CS152 (SEQ ID NO:208) encode full lengthpolypeptides having the amino acid sequences set forth in SEQ IDNOS:140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176,179, 182, 185, 188, 191, 194, 197, 200, 203, 206, and 209, which whenprocessed yield mature polypeptides having the amino acid sequences setforth in SEQ ID NOS:141, 144, 147, 150, 153, 156, 159, 162, 165, 168,171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, and210, respectively.

Group 18 comprises five Picramnia pentandra sequences of which CS161(SEQ ID NO:211) is the representative sequence. CS161 (SEQ ID NO:211),CS164 (SEQ ID NO:214), CS160 (SEQ ID NO:217), CS162 (SEQ ID NO:220), andCS163 (SEQ ID NO:223) encode full length polypeptides having the aminoacid sequences set forth in SEQ ID NOS:212, 215, 218, 221, and 224,which when processed yield mature polypeptides having the amino acidsequences set forth in SEQ ID NOS:213, 216, 219, 222, and 225,respectively.

Group 19 comprises three Nicotiana benthamiana sequences of which CS165(SEQ ID NO:226) is the representative sequence. CS165 (SEQ ID NO:226),CS168 (SEQ ID NO:229), and CS169 (SEQ ID NO:232) encode full lengthpolypeptides having the amino acid sequences set forth in SEQ IDNOS:227, 230, and 233, which when processed yield mature polypeptideshaving the amino acid sequences set forth in SEQ ID NOS:228, 231, and234, respectively.

Group 20 comprises CS003 (SEQ ID NO:235, PDF3), a maize gene. The fulllength CS003 polypeptide (SEQ ID NO:236) is expected to have a signalpeptide, thus the mature protein (SEQ ID NO:237) is likely to besecreted or extracellular.

Group 21 comprises CS007 (SEQ ID NO:238, PDF7), a maize gene. PDF7 islikely to be expressed in a kernel-specific or kernel-preferredexpression pattern. The full length CS007 polypeptide (SEQ ID NO:239) isexpected to have a signal peptide, thus the mature protein (SEQ IDNO:240) would be extracellular.

Group 22 comprises CS008 (SEQ ID NO:241, PDF8), a maize gene. PDF8 isexpressed in a kernel-preferred expression pattern. The full lengthCS008 polypeptide (SEQ ID NO:242) is expected to have a signal peptide,thus the mature protein (SEQ ID NO:243) would be extracellular. PDF8 maybe secreted.

Group 23 comprises CS009 (SEQ ID NO:244, PDF9), a maize gene. PDF9 isexpressed in a kernel-preferred pattern, particularly an endosperm- andpericarp-preferred expression pattern. The full length CS009 polypeptide(SEQ ID NO:245) includes a predicted transit peptide, thus the matureprotein (SEQ ID NO:246) would be extracellular.

Group 24 comprises CS010 (SEQ ID NO:247, PDF10), a maize gene. The fulllength CS010 polypeptide (SEQ ID NO:248) is expected to have a signalpeptide, thus the mature protein (SEQ ID NO:249) is likely to besecreted or extracellular. Expression appears to be kernel-specific orkernel-preferred.

Group 25 comprises CS014 (SEQ ID NO:250, PDF14), a maize gene. PDF14 isexpressed in a tassel-preferred manner. The full length CS014polypeptide (SEQ ID NO:251) appears to include a signal peptide, thusthe mature protein (SEQ ID NO:252) would be secreted.

Group 26 comprises CS016 (SEQ ID NO:253, PDF16), a maize gene. The PDF16nucleotide sequence appears to be expressed preferentially in thekernel, particularly in the endosperm and scutellum. The full lengthCS016 polypeptide (SEQ ID NO:254) is predicted to have a signal peptideending at amino acid position 22, thus the mature protein (SEQ IDNO:255) is likely to be an extracellular polypeptide.

Group 27 comprises CS019 (SEQ ID NO:256, PDF19), a maize gene.Expression of the sequence appears to be kernel-preferred. The proteinis likely extracellular as the full length CS019 polypeptide (SEQ IDNO:257) contains a signal peptide, thus the mature protein (SEQ IDNO:258) appears to be secreted or extracellular.

Group 28 comprises CS020 (SEQ ID NO:259, PDF20), a maize gene.Expression of PDF20 appears to be kernel-preferred. The predictedprotein appears to be extracellular as it has a predicted signalpeptide. The full length CS020 polypeptide (SEQ ID NO:260) is expectedto have a signal peptide, thus the mature protein (SEQ ID NO:261) islikely to be secreted or extracellular.

Group 29 comprises CS043 (SEQ ID NO:262), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:263, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:264.

Group 30 comprises CS045 (SEQ ID NO:265), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:266, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:267.

Group 31 comprises CS046 (SEQ ID NO:268), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:269, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:270.

Group 32 comprises CS048 (SEQ ID NO:271), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:272, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:273.

Group 33 comprises CS049 (SEQ ID NO:274), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:275, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:276.

Group 34 comprises CS060 (SEQ ID NO:277), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:278, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:279.

Group 35 comprises CS061 (SEQ ID NO:280), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:281, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:282.

Group 36 comprises CS068 (SEQ ID NO:283), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:284, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:285.

Group 37 comprises CS071 (SEQ ID NO:286), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:287, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:288.

Group 38 comprises CS072 (SEQ ID NO:289), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:290, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:291.

Group 39 comprises CS076 (SEQ ID NO:292), a Beta vulgaris sequence. Thisnucleotide sequence encodes a full length polypeptide having the aminoacid sequence set forth in SEQ ID NO:293, which when processed yields amature polypeptide having the amino acid sequence set forth in SEQ IDNO:294.

Group 40 comprises CS085 (SEQ ID NO:295), a Hedera helix sequence. Thisnucleotide sequence encodes a full length polypeptide having the aminoacid sequence set forth in SEQ ID NO:296, which when processed yields amature polypeptide having the amino acid sequence set forth in SEQ IDNO:297.

Group 41 comprises CS103 (SEQ ID NO:298), a Tulipa gesneriana sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:299, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:300.

Group 42 comprises CS124 (SEQ ID NO:301), an Amaranthus retroflexussequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:302, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:303.

Group 43 comprises CS159 (SEQ ID NO:304), an Allium porrum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:305, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:306.

Group 44 comprises CS113 (SEQ ID NO:307), a Nicotiana benthamianasequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:308, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:309.

Group 45 comprises CS095 (SEQ ID NO:310), a Tulipa gesneriana sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:311, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:312.

Group 46 comprises CS077 (SEQ ID NO:313), a Beta vulgaris sequence. Thisnucleotide sequence encodes a full length polypeptide having the aminoacid sequence set forth in SEQ ID NO:314, which when processed yields amature polypeptide having the amino acid sequence set forth in SEQ IDNO:315.

Group 47 comprises three Cyamopsis tetragonoloba sequences of whichCS108 (SEQ ID NO:316) is the representative sequence. CS108 (SEQ IDNO:316), CS156 (SEQ ID NO:319), and CS157 (SEQ ID NO:322) encode fulllength polypeptides having the amino acid sequences set forth in SEQ IDNOS:317, 320, and 323, which when processed yield mature polypeptideshaving the amino acid sequences set forth in SEQ ID NOS:318, 321, and324, respectively.

Group 48 comprises three sequences of which CS005 (SEQ ID NO:325, PDF5)is the representative sequence. CS005 was isolated from Zea mays. ThePDF5 expression pattern suggests fairly wide-distribution of expression.However, there is strong representation of CS005 in endosperms andembryos of kernels. The mature peptide appears to be extracellular, as asignal peptide is included in the sequence. CS005 (SEQ ID NO:325), CS042(SEQ ID NO:328) and CS067 (SEQ ID NO:331) encode full lengthpolypeptides having the amino acid sequences set forth in SEQ IDNOS:326, 329, and 332, which when processed yield mature polypeptideshaving the amino acid sequences set forth in SEQ ID NOS:327, 330, and333, respectively. CS042 and CS067 were isolated from Triticum aestivum.

Group 49 comprises four sequences of which CS053 (SEQ ID NO:337) is therepresentative sequence. CS100 (SEQ ID NO:334), CS053 (SEQ ID NO:337),CS064 (SEQ ID NO:340), and CS096 (SEQ ID NO:343) encode full lengthpolypeptides having the amino acid sequences set forth in SEQ IDNOS:335, 338, 341, and 344, which when processed yield maturepolypeptides having the amino acid sequences set forth in SEQ IDNOS:336, 339, 342, and 345, respectively. CS100 and CS096 were isolatedfrom Tulipa gesneriana. CS053 and CS064 were isolated from Triticumaestivum.

Group 50 comprises CS036 (SEQ ID NO:346), a Glycine max sequence. Thisnucleotide sequence encodes a full length polypeptide having the aminoacid sequence set forth in SEQ ID NO:347, which when processed yields amature polypeptide having the amino acid sequence set forth in SEQ IDNO:348.

Group 51 comprises CS125 (SEQ ID NO:349), a Brassica napus sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:350, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:351.

Group 52 comprises CS056 (SEQ ID NO:352), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:353, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:354.

Group 53 comprises CS047 (SEQ ID NO:355), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:356, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:357.

Group 54 comprises CS021 (SEQ ID NO:358, PDF21), a Zea mays sequence.PDF21 may be predominately expressed in the kernel. The sequence appearsto be extracellular as it includes a signal peptide. This nucleotidesequence encodes a full length polypeptide having the amino acidsequence set forth in SEQ ID NO:359, which when processed yields amature polypeptide having the amino acid sequence set forth in SEQ IDNO:360.

Group 55 comprises CS122 (SEQ ID NO:361), a Vernonia mespilifoliasequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:362, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:363.

Group 56 comprises CS111 (SEQ ID NO:364), a Picramnia pentandrasequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:365, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:366.

Group 57 comprises CS171 (SEQ ID NO:367), a Vernonia mespilifoliasequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:368, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:369.

Group 58 comprises CS172 (SEQ ID NO:370), a Vernonia mespilifoliasequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:371, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:372.

Group 59 comprises CS030 (SEQ ID NO:373), an Oryza sativa sequence. Thisnucleotide sequence encodes a full length polypeptide having the aminoacid sequence set forth in SEQ ID NO:374, which when processed yields amature polypeptide having the amino acid sequence set forth in SEQ IDNO:375.

Group 60 comprises CS088 (SEQ ID NO:376), a Parthenium argentatumsequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:377, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:378.

Group 61 comprises CS107 (SEQ ID NO:379), a Cyamopsis tetragonolobasequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:380, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:381.

Group 62 comprises CS058 (SEQ ID NO:382), a Triticum aestivum sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:383, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:384.

Group 63 comprises CS037 (SEQ ID NO:385), a Glycine max sequence. Thisnucleotide sequence encodes a full length polypeptide having the aminoacid sequence set forth in SEQ ID NO:386, which when processed yields amature polypeptide having the amino acid sequence set forth in SEQ IDNO:387.

Group 64 comprises two sequences of which CS082 (SEQ ID NO:391) is therepresentative sequence. CS086 (SEQ ID NO:388) and CS082 (SEQ ID NO:391)encode full length polypeptides having the amino acid sequences setforth in SEQ ID NOS:389 and 392, which when processed yield maturepolypeptides having the amino acid sequences set forth in SEQ ID NOS:390and 393, respectively. CS086 was isolated from Licania michauxii, andCS082 was isolated from Chrysobalanus icaco.

Group 65 comprises CS081 (SEQ ID NO:394), a Ricinus communis sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:395, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:396.

Group 66 comprises two Vernonia mespilifolia sequences of which CS121(SEQ ID NO:400) is the representative sequence. CS123 (SEQ ID NO:397)and CS121 (SEQ ID NO:400) encode full length polypeptides having theamino acid sequences set forth in SEQ ID NOS:398 and 401, which whenprocessed yield mature polypeptides having the amino acid sequences setforth in SEQ ID NOS:399 and 402, respectively.

Group 67 comprises CS080 (SEQ ID NO:403), a Ricinus communis sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:404, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:405.

Group 68 comprises two sequences of which CS083 (SEQ ID NO:406) is therepresentative sequence. CS083 (SEQ ID NO:406) and CS087 (SEQ ID NO:409)encode full length polypeptides having the amino acid sequences setforth in SEQ ID NOS:407 and 410, which when processed yield maturepolypeptides having the amino acid sequences set forth in SEQ ID NOS:408and 411, respectively. CS083 was isolated from Eucalyptus grandis, andCS087 was isolated from Tropaeolum majus.

Group 69 comprises CS155 (SEQ ID NO:412), a Cyamopsis tetragonolobasequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:413, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:414.

Group 70 comprises three Vitis vinifera sequences of which CS117 (SEQ IDNO:421) is the representative sequence. CS119 (SEQ ID NO:415), CS116(SEQ ID NO:418), and CS117 (SEQ ID NO:421) encode full lengthpolypeptides having the amino acid sequences set forth in SEQ IDNOS:416, 419, and 422, which when processed yield mature polypeptideshaving the amino acid sequences set forth in SEQ ID NOS:417, 420, and423, respectively.

Group 71 comprises CS126 (SEQ ID NO:424), a Eucalyptus grandis sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:425, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:426.

Group 72 comprises CS109 (SEQ ID NO:427), a Cyamopsis tetragonolobasequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:428, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:429.

Group 73 comprises CS115 (SEQ ID NO:430), a Nicotiana benthamianasequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:431, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:432.

Group 74 comprises CS089 (SEQ ID NO:433), a Parthenium argentatumsequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:434, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:435.

Group 75 comprises CS110 (SEQ ID NO:436), a Cyamopsis tetragonolobasequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:437, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:438.

Group 76 comprises CS158 (SEQ ID NO:439), a Cyamopsis tetragonolobasequence. This nucleotide sequence encodes a full length polypeptidehaving the amino acid sequence set forth in SEQ ID NO:440, which whenprocessed yields a mature polypeptide having the amino acid sequence setforth in SEQ ID NO:441.

Group 77 comprises CS127 (SEQ ID NO:442), a Licania michauxii sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:443, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:444.

Group 78 comprises two Vitis vinifera. sequences of which CS118 (SEQ IDNO:445) is the representative sequence. CS118 (SEQ ID NO:445) and CS170(SEQ ID NO:448) encode full length polypeptides having the amino acidsequences set forth in SEQ ID NOS:446 and 449, which when processedyield mature polypeptides having the amino acid sequences set forth inSEQ ID NOS:447 and 450, respectively.

Group 79 comprises CS090 (SEQ ID NO:451), an Arachis hypogaea sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:452, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:453.

Group 80 comprises CS075 (SEQ ID NO:454), a Brassica napus. sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:455, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:456.

Group 81 comprises CS011 (SEQ ID NO:457, PDF11), a Zea mays sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:458, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:459. PDF11 expression seems to be tassel-preferred. The maturepeptide may be secreted or extracellular as the sequence includes asignal peptide.

Group 82 comprises CS012 (SEQ ID NO:460, PDF12), a Zea mays sequence.This nucleotide sequence encodes a full length polypeptide having theamino acid sequence set forth in SEQ ID NO:461, which when processedyields a mature polypeptide having the amino acid sequence set forth inSEQ ID NO:462. The sequence may be preferentially expressed in kernels.PDF12 encodes a signal peptide, and thus would be secreted.

Group 83 comprises CS002 (SEQ ID NO:463, PDF2), a Zea mays sequence.This nucleotide sequence encodes a full-length polypeptide having theamino acid sequence set forth in SEQ ID NO:464. PDF2 is expressed in akernel-preferred pattern.

Group 84 comprises CS015 (SEQ ID NO:465, PDF15), a Zea mays sequence.This nucleotide sequence encodes a full-length polypeptide having theamino acid sequence set forth in SEQ ID NO:466. PDF15 is preferentiallyexpressed in kernels. The Zm-PDF15 sequence differs from Zm-ES-4 (Cordtset al. (2001) Plant J. 25:103-114) by one amino acid residue. The geneappears to encode an extracellularly localized protein as there is asignal peptide.

Group 85 comprises CS001 (SEQ ID NO:467, PDF1), a Zea mays sequence.This nucleotide sequence encodes a full-length polypeptide having theamino acid sequence set forth in SEQ ID NO:468. Zm-PDF1 is predicted tobe an extracellular protein.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. An “isolated” or “purified” nucleic acidmolecule or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the nucleic acid molecule or protein as foundin its naturally occurring environment. Thus, an isolated or purifiednucleic acid molecule or protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Preferably, an “isolated” nucleic acid is freeof sequences (preferably protein encoding sequences) that naturallyflank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends ofthe nucleic acid) in the genomic DNA of the organism from which thenucleic acid is derived. For example, in various embodiments, theisolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, preferably culture medium represents lessthan about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemicalprecursors or non-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence havedefensin-like activity and thereby affect development, developmentalpathways, and defense responses. Alternatively, fragments of anucleotide sequence that are useful as hybridization probes generally donot encode fragment proteins retaining biological activity. Thus,fragments of a nucleotide sequence may range from at least about 20nucleotides, about 50 nucleotides, about 100 nucleotides, and up to thefull-length nucleotide sequence encoding the proteins of the invention.

A fragment of a defensin nucleotide sequence that encodes a biologicallyactive portion of a defensin protein of the invention will encode atleast 15, 25, 30, 50, 100, 150, 153, 200, 250, 300 contiguous aminoacids, or up to the total number of amino acids present in a full-lengthprotein of the invention. Fragments of a defensin nucleotide sequencethat are useful as hybridization probes for PCR primers generally neednot encode a biologically active portion of a defensin protein.

Thus, a fragment of a defensin nucleotide sequence may encode abiologically active portion of a defensin protein, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of a defensinprotein can be prepared by isolating a portion of one of the defensinnucleotide sequences of the invention, expressing the encoded portion ofthe defensin protein (e.g., by recombinant expression in vitro), andassessing the activity of the encoded portion of the defensin protein.Nucleic acid molecules that are fragments of a defensin nucleotidesequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 683, 700, 800, or 900 nucleotides, or upto the number of nucleotides present in a full-length defensinnucleotide sequence disclosed herein

By “variants” substantially similar sequences are intended. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the defensin polypeptides of the invention.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis but which still encode adefensin protein of the invention. Generally, variants of a particularnucleotide sequence of the invention will have at least about 50%, 60%,65%, 70%, generally at least about 75%, 80%, 85%, preferably at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably atleast about 98%, 99% or more sequence identity to that particularnucleotide sequence as determined by sequence alignment programsdescribed elsewhere herein using default parameters.

By “variant protein” a protein derived from the native protein bydeletion (so-called truncation) or addition of one or more amino acidsto the N-terminal and/or C-terminal end of the native protein; deletionor addition of one or more amino acids at one or more sites in thenative protein; or substitution of one or more amino acids at one ormore sites in the native protein is intended. Variant proteinsencompassed by the present invention are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, defensin-like activity as described herein. Suchvariants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a native defensinprotein of the invention will have at least about 40%, 50%, 60%, 65%,70%, generally at least about 75%, 80%, 85%, preferably at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at leastabout 98%, 99% or more sequence identity to the amino acid sequence forthe native protein as determined by sequence alignment programsdescribed elsewhere herein using default parameters. A biologicallyactive variant of a protein of the invention may differ from thatprotein by as few as 1-15 amino acid residues, as few as 1-10, such as6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

Biological activity of the defensin polypeptides (i.e., influencing theplant defense response and various developmental pathways, including,for example, influencing cell division) can be assayed by any methodknown in the art. Biological activity of the polypeptides of the presentinvention can be assayed by any method known in the art (see forexample, U.S. Pat. No. 5,614,395; Thomma et al. (1998) Plant Biology95:15107-15111; Liu et al. (1994) Plant Biology 91:1888-1892; Hu et al.(1997) Plant Mol. Biol. 34:949-959; Cammue et al. (1992) J. Biol. Chem.267:2228-2233; and Thevissen et al. (1996) J. Biol. Chem.271:15018-15025, all of which are herein incorporated by reference).Furthermore, assays to detect defensin-like activity include, forexample, assessing antifungal and/or antimicrobial activity (Terras etal. (1992) J. Biol. Chem. 267:14301-15309; Terras et al. (1993) PlantPhysiol (Bethesda) 103:1311-1319; Terras et al. (1995) Plant Cell7:573-588, Moreno et al. (1994) Eur. J. Biochem. 223:135-139; and Osbornet al. (1995) FEBS Lett. 368:257-262, all of which are hereinincorporated by reference).

The polypeptides of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Novel proteins having properties of interest may be createdby combining elements and fragments of proteins of the present inventionas well as other proteins. Methods for such manipulations are generallyknown in the art. For example, amino acid sequence variants of thedefensin proteins can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (Macmillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be preferred.

Thus, the genes and nucleotide sequences of the invention include boththe naturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass naturally occurring proteins as wellas variations and modified forms thereof. Such variants will continue topossess the desired developmental activity, or defense responseactivity. Obviously, the mutations that will be made in the DNA encodingthe variant must not place the sequence out of reading frame andpreferably will not create complementary regions that could producesecondary mRNA structure. See, EP Patent Application Publication No.75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by defensin activity assays. See, for example, Lancaster etal. (1994) J. Biol. Chem. 14:1137-1142 and Terras et al. (1995) PlantCell 7:537-588, herein incorporated by reference. Additionally,differences in the expression of specific genes between uninfected andinfected plants can be determined using gene expression profiling. RNAwas analyzed using the gene expression profiling process (GeneCalling®)as described in U.S. Pat. No. 5,871,697, herein incorporated byreference.

Variant nucleotide sequences and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different defensincoding sequences can be manipulated to create a new defensin proteinpossessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants.In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences based on their sequence homology to thesequences set forth herein. Sequences isolated based on their sequenceidentity to the entire defensin sequences set forth herein or tofragments thereof are encompassed by the present invention. Suchsequences include sequences that are orthologs of the disclosedsequences. By “orthologs” genes derived from a common ancestral gene andwhich are found in different species as a result of speciation isintended. Genes found in different species are considered orthologs whentheir nucleotide sequences and/or their encoded protein sequences sharesubstantial identity as defined elsewhere herein. Functions of orthologsare often highly conserved among species. Thus, isolated sequences thatencode a defensin and which hybridize under stringent conditions to thedefensin sequences disclosed herein, or to fragments thereof, areencompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the defensin sequences ofthe invention. Methods for preparation of probes for hybridization andfor construction of cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

For example, an entire defensin sequence disclosed herein, or one ormore portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding defensin sequences and messenger RNAs. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique among defensin sequences andare preferably at least about 10 nucleotides in length, and mostpreferably at least about 20 nucleotides in length. Such probes may beused to amplify corresponding sequences from a chosen organism by PCR.This technique may be used to isolate additional coding sequences from adesired organism or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y. ).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” conditions under which a probe will hybridize to its targetsequence to a detectably greater degree than to other sequences (e.g.,at least 2-fold over background) is intended. Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% form)−500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The T_(m) is the temperature (under defined ionic strength andpH) at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with ≧90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the thermal melting point (T_(m)) for the specific sequenceand its complement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

Thus, isolated sequences that encode a defensin polypeptide and whichhybridize under stringent conditions to the defensin sequences disclosedherein, or to fragments thereof, are encompassed by the presentinvention.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al. (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seehttp://www.ncbi.hlm.nih.gov. Alignment may also be performed manually byinspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity using GAP Weight of 50 and LengthWeight of 3; % similarity using Gap Weight of 12 and Length Weight of 4,or any equivalent program. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by the preferred program.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least90%, and most preferably at least 95%, compared to a reference sequenceusing one of the alignment programs described using standard parameters.One of skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, more preferably atleast 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C., depending uponthe desired degree of stringency as otherwise qualified herein. Nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70% sequenceidentity to a reference sequence, preferably 80%, more preferably 85%,most preferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman et al. (1970) J. Mol. Biol. 48:443. An indication that twopeptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

Disease and Pests

Compositions and methods for controlling pathogenic agents are provided.The anti-pathogenic compositions comprise plant defensin nucleotide andamino acid sequences. Particularly, the plant nucleic acid and aminoacid sequences and fragments and variants thereof set forth hereinpossess anti-pathogenic activity. Accordingly, the compositions andmethods are useful in protecting plants against fungal pathogens,viruses, nematodes, insects, and the like. Additionally provided aretransformed plants, plant cells, plant tissues and seeds thereof.

By “plant pathogen” or “plant pest” any organism that can cause harm toa plant, by inhibiting or slowing the growth of a plant, by damaging thetissues of a plant, by weakening the immune system of a plant, reducingthe resistance of a plant to abiotic stresses, and/or by causing thepremature death of the plant, etc. is intended. Plant pathogens andplant pests include insects, nematodes, and organisms such as fungi,viruses, and bacteria.

By “disease resistance” or “pathogen resistance” it is intended that theorganisms avoid the disease symptoms which are the outcome oforganism-pathogen interactions. That is, pathogens are prevented fromcausing diseases and the associated disease symptoms, or alternatively,the disease symptoms caused by the pathogen is minimized or lessened.

By “anti-pathogenic compositions” is intended that the compositions ofthe invention are capable of suppressing, controlling, and/or killingthe invading pathogenic organism. An antipathogenic composition of theinvention will reduce the disease symptoms resulting from pathogenchallenge by at least about 5% to about 50%, at least about 10% to about60%, at least about 30% to about 70%, at least about 40% to about 80%,or at least about 50% to about 90% or greater. Hence, the methods of theinvention can be utilized to protect plants from disease, particularlythose diseases that are caused by plant pathogens.

An “antimicrobial agent,” a “pesticidal agent,” a “defensin,” an“antiviral agent,” and “insecticidal agent,” and/or a “fungicidal agent”will act similarly to suppress, control, and/or kill the invadingpathogen.

A defensive agent will possess defensive activity. By “defensiveactivity” an antipathogenic, antimicrobial, antiviral, insecticidal, orantifungal activity is intended.

By “antipathogenic compositions” it is intended that the compositions ofthe invention have activity against pathogens; including fungi,microorganisms, viruses, insects and nematodes, and thus are capable ofsuppressing, controlling, and/or killing the invading pathogenicorganism. An antipathogenic composition of the invention will reduce thedisease symptoms resulting from pathogen challenge by at least about 5%to about 50%, at least about 10% to about 60%, at least about 30% toabout 70%, at least about 40% to about 80%, or at least about 50% toabout 90% or greater. Hence, the methods of the invention can beutilized to protect organisms, particularly plants, from disease,particularly those diseases that are caused by invading pathogens.

Assays that measure antipathogenic activity are commonly known in theart, as are methods to quantitate disease resistance in plants followingpathogen infection. See, for example, U.S. Pat. No. 5,614,395, hereinincorporated by reference. Such techniques include, measuring over time,the average lesion diameter, the pathogen biomass, and the overallpercentage of decayed plant tissues. For example, a plant eitherexpressing an antipathogenic polypeptide or having an antipathogeniccomposition applied to its surface shows a decrease in tissue necrosis(i.e., lesion diameter) or a decrease in plant death following pathogenchallenge when compared to a control plant that was not exposed to theantipathogenic composition. Alternatively, antipathogenic activity canbe measured by a decrease in pathogen biomass. For example, a plantexpressing an antipathogenic polypeptide or exposed to an antipathogeniccomposition is challenged with a pathogen of interest. Over time, tissuesamples from the pathogen-inoculated tissues are obtained and RNA isextracted. The percent of a specific pathogen RNA transcript relative tothe level of a plant specific transcript allows the level of pathogenbiomass to be determined. See, for example, Thomma et al. (1998) PlantBiology 95:15107-15111, herein incorporated by reference.

Furthermore, in vitro antipathogenic assays include, for example, theaddition of varying concentrations of the antipathogenic composition topaper disks and placing the disks on agar containing a suspension of thepathogen of interest. Following incubation, clear inhibition zonesdevelop around the discs that contain an effective concentration of theantipathogenic polypeptide (Liu et al. (1994) Plant Biology91:1888-1892, herein incorporated by reference). Additionally,microspectrophotometrical analysis can be used to measure the in vitroantipathogenic properties of a composition (Hu et al. (1997) Plant Mol.Biol. 34:949-959 and Cammue et al. (1992) J. Biol. Chem. 267:2228-2233,both of which are herein incorporated by reference).

In specific embodiments, methods for increasing pathogen resistance in aplant comprise stably transforming a plant with a DNA constructcomprising an anti-pathogenic nucleotide sequence of the inventionoperably linked to promoter that drives expression in a plant. Suchmethods find use in agriculture particularly in limiting the impact ofplant pathogens on crop plants. While the choice of promoter will dependon the desired timing and location of expression of the anti-pathogenicnucleotide sequences, preferred promoters include constitutive andpathogen-inducible promoters.

It is understood in the art that plant DNA viruses and fungal pathogensremodel the control of the host replication and gene expressionmachinery to accomplish their own replication and effective infection.The present invention may be useful in preventing such corruption of thecell.

The defensin sequences find use in disrupting cellular function of plantpathogens or insect pests as well as altering the defense mechanisms ofa host plant to enhance resistance to disease or insect pests. While theinvention is not bound by any particular mechanism of action to enhancedisease resistance, the gene products of the defensin sequences functionto inhibit or prevent diseases in a plant.

The methods of the invention can be used with other methods available inthe art for enhancing disease resistance in plants. For example, any oneof a variety of second nucleotide sequences may be utilized, embodimentsof the invention encompass those second nucleotide sequences that, whenexpressed in a plant, help to increase the resistance of a plant topathogens. It is recognized that such second nucleotide sequences may beused in either the sense or antisense orientation depending on thedesired outcome. Other plant defense proteins include those described inPCT patent publications WO 99/43823 and WO 99/43821, both of which areherein incorporated by reference.

Pathogens of the invention include, but are not limited to, viruses orviroids, bacteria, insects, nematodes, fungi, and the like. Virusesinclude any plant virus, for example, tobacco or cucumber mosaic virus,ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Specificfungal and viral pathogens for the major crops include: Soybeans:Phytophthora megasperma f.sp. glycinea, Macrophomina phaseolina,Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum,Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthephaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii,Cercospora sojina, Peronospora manshurica, Colletotrichum dematium(Colletotrichum truncatum), Corynespora cassiicola, Septoria glycines,Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v.glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa,Fusarium semitectum, Phialophora gregata, Soybean mosaic virus,Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus,Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythiumdebaryanum, Tomato spotted wilt virus, Heterodera glycines, Fusariumsolani; Canola: Albugo candida, Alternaria brassicae, Leptosphaeriamaculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerellabrassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum,Alternaria alternata; Alfalfa: Clavibacter Michigan's subsp. insidiosum,Pythium ultimum, Pythium irregulare, Pythium splendens, Pythiumdebaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronosporatrifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis,Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium spp.,Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches, Stemphyliumherbarum, Stemphylium alfalfae; Wheat: Pseudomonas syringae p.v.atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v.translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata,Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum,Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporiumgramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici,Puccinia graminis f.sp. tritici, Puccinia recondita f.sp. tritici,Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum,Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides,Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var.tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum,Bipolaris sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus,Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat SpindleStreak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletiatritici, Tilletia laevis, Tilletia indica, Pythium gramicola, HighPlains Virus, European wheat striate virus; Sunflower: Plasmophorahalstedii, Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi,Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytiscinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphecichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer,Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum p.v.carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugotragopogonis; Corn: Fusarium moniliforme var. subglutinans, Erwiniastewartii, Fusarium moniliforme, Gibberella zeae (Fusarium graminearum),Stenocarpella maydis (Diplodia maydis), Pythium irregulare, Pythiumdebaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum,Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T(Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III(Cochliobolus carbonum), Exserohilum turcicum I, II & III,Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis,Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi,Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum,Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvulariainaequalis, Curvularia pallescens, Clavibacter michiganense subsp.nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, WheatStreak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi,Pseudomonas avenae, Erwinia chrysanthemi p.v. zea, Erwinia carotovora,Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora,Peronosclerospora sorghi, Peronosclerospora philippinensis,Peronosclerospora maydis, Peronosclerospora saccharin, Sphacelothecareiliana, Physopella zeae, Cephalosporium maydis, Cephalosporiumacremonium, Maize Chlorotic Mottle Virus, High Plains Virus, MaizeMosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize StripeVirus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum,Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi,Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v.syringae, Xanthomonas campestris p.v. holcicola, Pseudomonasandropogonis, Puccinia purpurea, Macrophomina phaseolina, Periconiacircinata, Fusarium moniliforme, Alternaria alternata, Bipolarissorghicola, Helminthosporium sorghicola, Curvularia lunata, Phomainsidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulisporasorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisoriumreilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisoriumsorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Clavicepssorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthonamacrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis,Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum,Pythium arrhenomanes, Pythium graminicola, etc.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera and Globodera spp.; particularlyGlobodera rostochiensis and Globodera pailida (potato cyst nematodes);Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beetcyst nematode); and Heterodera avenae (cereal cyst nematode). Additionalnematodes include: Heterodera cajani; Heterodera trifolii; Heteroderaoryzae; Globodera tabacum; Meloidogyne incognita; Meloidogyne javonica;Meloidogyne hapla; Meloidogyne arenaria; Meloidogyne naasi; Meloidogyneexigua; Xiphinema index; Xiphinema italiae; Xiphinema americanum;Xiphinema diversicaudatum; Pratylenchus penetrans; Pratylenchusbrachyurus; Pratylenchus zeae; Pratylenchus coffeae; Pratylenchusthornei; Pratylenchus scribneri; Pratylenchus vulnus; Pratylenchuscurvitatus; Radopholus similis; Radopholus citrophilus; Ditylenchusdipsaci; Helicotylenchus multicintus; Rotylenchulus reniformis;Belonolaimus spp.; Paratrichodorus anemones; Trichodorus spp.;Primitivus spp.; Anguina tritici; Bider avenae; Subanguina radicicola;Tylenchorhynchus spp.; Haplolaimus seinhorsti; Tylenchulussemipenetrans; Hemicycliophora arenaria; Belonolaimus langicaudatus;Paratrichodorus xiphinema; Paratrichodorus christiei; Rhadinaphelenchuscocophilus; Paratrichodorus minor; Hoplolaimus galeatus; Hoplolaimuscolumbus; Criconemella spp.; Paratylenchus spp.; Nacoabbus aberrans;Aphelenchoides besseyi; Ditylenchus angustus; Hirchmaniella spp.;Scutellonema spp.; Hemicriconemoides kanayaensis; Tylenchorynchusclaytoni; and Cacopaurus pestis.

Insect pests include insects selected from the orders Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., particularly Coleoptera and Lepidoptera. Insect pestsof the invention for the major crops include: Maize: Ostrinia nubilalis,European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea,corn earworm; Spodoptera frugiperda, fall armyworm; Diatraeagrandiosella, southwestern corn borer; Elasmopalpus lignosellus, lessercornstalk borer; Diatraea saccharalis, sugarcane borer; Diabroticavirgifera, western corn rootworm; Diabrotica longicornis barberi,northern corn rootworm; Diabrotica undecimpunctata howardi, southerncorn rootworm; Melanotus spp., wireworms; Cyclocephala borealis,northern masked chafer (white grub); Cyclocephala immaculata, southernmasked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blotch leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; Zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia spp., Rootmaggots.

Expression of Sequences

The nucleic acid sequences of the present invention can be expressed ina host cell such as bacterial, fungal, yeast, insect, mammalian, orpreferably plant cells. It is expected that those of skill in the artare knowledgeable in the numerous expression systems available forexpression of a nucleic acid encoding a protein of the presentinvention. No attempt to describe in detail the various methods knownfor the expression of proteins in prokaryotes or eukaryotes will bemade.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous nucleotidesequence can be from a species different from that from which thenucleotide sequence was derived, or, if from the same species, thepromoter is not naturally found operably linked to the nucleotidesequence. A heterologous protein may originate from a foreign species,or, if from the same species, is substantially modified from itsoriginal form by deliberate human intervention.

By “host cell” a cell, which comprises a heterologous nucleic acidsequence of the invention is meant. Host cells may be prokaryotic cellssuch as E. coli, or eukaryotic cells such as yeast, insect, amphibian,or mammalian cells. Preferably, host cells are monocotyledonous ordicotyledonous plant cells. A particularly preferred monocotyledonoushost cell is a maize host cell.

The defensin sequences of the invention are provided in expressioncassettes or DNA constructs for expression in the plant of interest. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa defensin sequence of the invention. By “operably linked” a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence is intended. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the defensin sequence to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, adefensin DNA sequence of the invention, and a transcriptional andtranslational termination region functional in plants. Thetranscriptional initiation region, the promoter, may be native oranalogous or foreign or heterologous to the plant host. Additionally,the promoter may be the natural sequence or alternatively a syntheticsequence. By “foreign” is intended that the transcriptional initiationregion is not found in the native plant into which the transcriptionalinitiation region is introduced. As used herein, a chimeric genecomprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

While it may be preferable to express the sequences using heterologouspromoters, the native promoter sequences may be used. Such constructswould change expression levels of defensin in the host cell (i.e., plantor plant cell). Thus, the phenotype of the host cell (i.e., plant orplant cell) is altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,or may be derived from another source. Convenient termination regionsare available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See alsoGuerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991)Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen etal. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.(1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed plant. That is, the genes can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, U.S. Pat. Nos.5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chainbinding protein (BiP), (Macejak et al. (1991) Nature 353:90-94);untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaicvirus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA,ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottlevirus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). Seealso, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Othermethods known to enhance translation can also be utilized, for example,introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glyphosate, glufosinate ammonium, bromoxynil, imidazolinones,and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992)Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff(1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724. Such disclosures are herein incorporatedby reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. That is, thenucleic acids can be combined with constitutive, tissue-preferred, orother promoters for expression in the host cell of interest. Suchconstitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, thosedisclosed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611, hereinincorporated by reference.

Generally, it will be beneficial to express the gene from an induciblepromoter, particularly from a pathogen-inducible promoter. Suchpromoters include those from pathogenesis-related proteins (PRproteins), which are induced following infection by a pathogen; e.g., PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, forexample, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Ukneset al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol.Virol. 4:111-116. See also WO 99/43819 published Sep. 9, 1999, hereinincorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant. Path. 41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2): 141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced defensinexpression within a particular plant tissue. Tissue-preferred promotersinclude Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al.(1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol GenGenet. 254(3):337-343; Russell et al. (1997) Transgenic Res.6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341;Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al.(1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant CellPhysiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; andGuevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

Leaf-specific promoters are known in the art. See, for example, Yamamotoet al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol.105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778;Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci.USA 90(20):9586-9590.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10: 108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphatesynthase); and celA (cellulose synthase) (see WO 00/11177, hereinincorporated by reference). Gama-zein is a preferred endosperm-specificpromoter. Glob-1 is a preferred embryo-specific promoter. For dicots,seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference.

The method of transformation/transfection is not critical to the instantinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied. Accordingly, a widevariety of methods have been developed to insert a DNA sequence into thegenome of a host cell to obtain the transcription and/or translation ofthe sequence to effect phenotypic changes in the organism. Thus, anymethod, which provides for effective transformation/transfection may beemployed.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particleacceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050;Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No.5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; McCabe et al. (1988)Biotechnology 6:923-926); and Lec1 transformation (WO 00/28058). Alsosee Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al.(1987) Particulate Science and Technology 5:27-37 (onion); Christou etal. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensurethat expression of the desired phenotypic characteristic has beenachieved.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp., Pisum spp.), and members of the genusCucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis),and musk melon (C. melo). Ornamentals include azalea (Rhododendronspp.), hydrangea (Hydrangea macrophylla), hibiscus (Hibiscusrosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthuscaryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Preferably, plants of the presentinvention are crop plants (for example, corn, alfalfa, sunflower,Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet,tobacco, etc.), more preferably corn and soybean plants, yet morepreferably corn plants.

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding sequences, include such commonly used promoters as thebeta lactamase (penicillinase) and lactose (lac) promoter systems (Changet al. (1977) Nature 198:1056), the tryptophan (trp) promoter system(Goeddel et al. (1980) Nucleic Acids Res. 8:4057) and the lambda derivedPL promoter and N-gene ribosome binding site (Simatake and Rosenberg(1981) Nature 292:128). Examples of selection markers for E. coliinclude, for example, genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al. (1983) Gene22:229-235 and Mosbach et al. (1983) Nature 302:543-545).

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a polynucleotide of the presentinvention can be expressed in these eukaryotic systems. In someembodiments, transformed/transfected plant cells, as discussed infra,are employed as expression systems for production of the proteins of theinstant invention. Such antimicrobial proteins can be used for anyapplication including coating surfaces to target microbes as describedfurther infra.

Synthesis of heterologous nucleotide sequences in yeast is well known.Sherman, F., et al. (1982) Methods in Yeast Genetics, Cold Spring HarborLaboratory is a well recognized work describing the various methodsavailable to produce proteins in yeast. Two widely utilized yeasts forproduction of eukaryotic proteins are Saccharomyces cerevisiae andPichia pastoris. Vectors, strains, and protocols for expression inSaccharomyces and Pichia are known in the art and available fromcommercial suppliers (e.g., Invitrogen). Suitable vectors usually haveexpression control sequences, such as promoters, including3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like, as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques, radioimmunoassay,or other standard immunoassay techniques.

The sequences of the present invention can also be ligated to variousexpression vectors for use in transfecting cell cultures of, forinstance, mammalian, insect, or plant origin. Illustrative cell culturesuseful for the production of the peptides are mammalian cells. A numberof suitable host cell lines capable of expressing intact proteins havebeen developed in the art, and include the HEK293, BHK21, and CHO celllines. Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter (e.g. the CMVpromoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter),an enhancer (Queen et al. (1986) Immunol. Rev. 89:49), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites (e.g., an SV40 large T Ag poly A additionsite), and transcriptional terminator sequences. Other animal cellsuseful for production of proteins of the present invention areavailable, for instance, from the American Type Culture Collection.

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See, Schneider(1987) J. Embyol. Exp. Morphol. 27:353-365).

As with yeast, when higher animal or plant host cells are employed,polyadenylation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague, et al.(1983) J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors. Saveria-Campo, M.,(1985) Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNACloning Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press,Arlington, Va. pp. 213-238.

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextrin, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art. Kuchler,R. J. (1997) Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc.

It is recognized that with these nucleotide sequences, antisenseconstructions, complementary to at least a portion of the messenger RNA(mRNA) for the defensin sequences can be constructed. Antisensenucleotides are constructed to hybridize with the corresponding mRNA.Modifications of the antisense sequences may be made as long as thesequences hybridize to and interfere with expression of thecorresponding mRNA. In this manner, antisense constructions having 70%,preferably 80%, more preferably 85% sequence identity to thecorresponding antisensed sequences may be used. Furthermore, portions ofthe antisense nucleotides may be used to disrupt the expression of thetarget gene. Generally, sequences of at least 50 nucleotides, 100nucleotides, 200 nucleotides, or greater may be used.

The nucleotide sequences of the present invention may also be used inthe sense orientation to suppress the expression of endogenous genes inplants. Methods for suppressing gene expression in plants usingnucleotide sequences in the sense orientation are known in the art. Themethods generally involve transforming plants with a DNA constructcomprising a promoter that drives expression in a plant operably linkedto at least a portion of a nucleotide sequence that corresponds to thetranscript of the endogenous gene. Typically, such a nucleotide sequencehas substantial sequence identity to the sequence of the transcript ofthe endogenous gene, preferably greater than about 65% sequenceidentity, more preferably greater than about 85% sequence identity, mostpreferably greater than about 95% sequence identity. See U.S. Pat. Nos.5,283,184 and 5,034,323; herein incorporated by reference.

In some embodiments, the content and/or composition of polypeptides ofthe present invention in a plant may be modulated by altering, in vivoor in vitro, the promoter of the nucleotide sequence to up- ordown-regulate expression. For instance, an isolated nucleic acidcomprising a promoter sequence operably linked to a polynucleotide ofthe present invention is transfected into a plant cell. Subsequently, aplant cell comprising the promoter operably linked to a polynucleotideof the present invention is selected for by means known to those ofskill in the art such as, but not limited to, Southern blot, DNAsequencing, or PCR analysis using primers specific to the promoter andto the gene and detecting amplicons produced therefrom. A plant or plantpart altered or modified by the foregoing embodiments is grown underplant forming conditions for a time sufficient to modulate theconcentration and/or composition of polypeptides of the presentinvention in the plant. Plant forming conditions are well known in theart and discussed briefly, supra. Detection of expression of apolypeptide of the invention occurs through any method known to one ofskill in the art including, but not limited to, immunolocalization.

In general, concentration or composition of the polypeptides of theinvention is increased or decreased by at least 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, or 90% relative to a native control plant, plantpart, or cell lacking the aforementioned recombinant expressioncassette. Modulation in the present invention may occur during and/orsubsequent to growth of the plant to the desired stage of development.Modulating nucleic acid expression temporally and/or in particulartissues can be controlled by employing the appropriate promoter operablylinked to a polynucleotide of the present invention in, for example,sense or antisense orientation as discussed in greater detail, supra.Induction of expression of a polynucleotide of the present invention canalso be controlled by exogenous administration of an effective amount ofinducing compound. Inducible promoters and inducing compounds, whichactivate expression from these promoters, are well known in the art. Invarious embodiments, the polypeptides of the present invention aremodulated in crop plants, particularly maize, wheat, soybean, alfalfa,barley, oats, and rice.

The methods of the invention can be used with other methods available inthe art for enhancing disease resistance in plants. Similarly, theantimicrobial compositions described herein may be used alone or incombination with other nucleotide sequences, polypeptides, or agents toprotect against plant diseases and pathogens. Although any one of avariety of second nucleotide sequences may be utilized, specificembodiments of the invention encompass those second nucleotide sequencesthat, when expressed in a plant, help to increase the resistance of aplant to pathogens.

Proteins, peptides, and lysozymes that naturally occur in insects(Jaynes et al. (1987) Bioassays 6:263-270), plants (Broekaert et al.(1997) Critical Reviews in Plant Sciences 16:297-323), animals (Vunnamet al. (1997) J. Peptide Res. 49:59-66), and humans (Mitra and Zang(1994) Plant Physiol. 106:977-981; Nakajima et al. (1997) Plant CellReports 16:674-679) are also a potential source of plant diseaseresistance. Examples of such plant resistance-conferring sequencesinclude those encoding sunflower rhoGTPase-Activating Protein (rhoGAP),lipoxygenase (LOX), Alcohol Dehydrogenase (ADH), andSclerotinia-Inducible Protein-1 (SCIP-1) described in U.S. applicationSer. No. 09/714,767, herein incorporated by reference. These nucleotidesequences enhance plant disease resistance through the modulation ofdevelopment, developmental pathways, and the plant pathogen defensesystem. Other plant defense proteins include those described in WO99/43823 and WO 99/43821, all of which are herein incorporated byreference. It is recognized that such second nucleotide sequences may beused in either the sense or antisense orientation depending on thedesired outcome.

In another embodiment, the defensins comprise isolated polypeptides ofthe invention. The defensins of the invention find use in thedecontamination of plant pathogens during the processing of grain foranimal or human food consumption; during the processing of feedstuffs,and during the processing of plant material for silage. In thisembodiment, the defensins of the invention are presented to grain, plantmaterial for silage, or a contaminated food crop, or during anappropriate stage of the processing procedure, in amounts effective forantimicrobial activity. The compositions can be applied to theenvironment of a plant pathogen by, for example, spraying, atomizing,dusting, scattering, coating or pouring, introducing into or on thesoil, introducing into irrigation water, by seed treatment, or dustingat a time when the plant pathogen has begun to appear or before theappearance of pests as a protective measure. It is recognized that anymeans that bring the defensive agent polypeptides in contact with theplant pathogen can be used in the practice of the invention.

Additionally, the compositions can be used in formulations used fortheir antimicrobial activities. Methods are provided for controllingplant pathogens comprising applying a decontaminating amount of apolypeptide or composition of the invention to the environment of theplant pathogen. The polypeptides of the invention can be formulated withan acceptable carrier into a composition(s) that is, for example, asuspension, a solution, an emulsion, a dusting powder, a dispersiblegranule, a wettable powder, an emulsifiable concentrate, an aerosol, animpregnated granule, an adjuvant, a coatable paste, and alsoencapsulations in, for example, polymer substances.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bacteriocides, nematocides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants, or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular targetmycotoxins. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g., natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders, or fertilizers. Theactive ingredients of the present invention are normally applied in theform of compositions and can be applied to the crop area or plant to betreated, simultaneously or in succession, with other compounds. In someembodiments, methods of applying an active ingredient of the presentinvention or an agrochemical composition of the present invention (whichcontains at least one of the proteins of the present invention) arefoliar application, seed coating, and soil application.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; a carboxylateof a long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate, or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g. polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as 2, 4, 7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate, oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include, but are not limited to, inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions of the present invention can be in a suitable form fordirect application or as concentrate of primary composition, whichrequires dilution with a suitable quantity of water or other diluentbefore application. The decontaminating concentration will varydepending upon the nature of the particular formulation, specifically,whether it is a concentrate or to be used directly.

In a further embodiment, the compositions, as well as the polypeptidesof the present invention can be treated prior to formulation to prolongthe activity when applied to the environment of a plant pathogen as longas the pretreatment is not deleterious to the activity. Such treatmentcan be by chemical and/or physical means as long as the treatment doesnot deleteriously affect the properties of the composition(s). Examplesof chemical reagents include, but are not limited to, halogenatingagents; aldehydes such as formaldehyde and glutaraldehyde;anti-infectives, such as zephiran chloride; alcohols, such asisopropanol and ethanol; and histological fixatives, such as Bouin'sfixative and Helly's fixative (see, for example, Humason (1967) AnimalTissue Techniques (W.H. Freeman and Co.)).

In an embodiment of the invention, the compositions of the inventioncomprise a microbe having stably integrated the nucleotide sequence of adefensive agent. The resulting microbes can be processed and used as amicrobial spray. Any suitable microorganism can be used for thispurpose. See, for example, Gaertner et al. (1993) in Advanced EngineeredPesticides, Kim (Ed.). In one embodiment, the nucleotide sequences ofthe invention are introduced into microorganisms that multiply on plants(epiphytes) to deliver the defensins to potential target crops.Epiphytes can be, for example, gram-positive or gram-negative bacteria.

It is further recognized that whole, i.e., unlysed, cells of thetransformed microorganism can be treated with reagents that prolong theactivity of the polypeptide produced in the microorganism when themicroorganism is applied to the environment of a target plant. Asecretion signal sequence may be used in combination with the gene ofinterest such that the resulting enzyme is secreted outside themicroorganism for presentation to the target plant.

In this manner, a gene encoding a defensive agent of the invention maybe introduced via a suitable vector into a microbial host, and saidtransformed host applied to the environment, plants, or animals.Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest may be selected for transformation. Thesemicroorganisms are selected so as to be capable of successfullycompeting in the particular environment with the wild-typemicroorganisms, to provide for stable maintenance and expression of thegene expressing the detoxifying polypeptide, and for improved protectionof the proteins of the invention from environmental degradation andinactivation.

Such microorganisms include bacteria, algae, and fungi. Illustrativeprokaryotes, both Gram-negative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae; and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like. Of particular interest are microorganisms,such as bacteria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella,Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius,Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g.,Saccharomyces, Pichia, Cryptococcus, Kluyveromyces, Sporobolomyces,Rhodotorula, Aureobasidium, and Gliocladium. Of particular interest aresuch phytosphere bacterial species as Pseudomonas syringae, Pseudomonasfluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria,Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti,Alcaligenes entrophus, Clavibacter xyli, and Azotobacter vinlandii; andphytosphere yeast species such as Rhodotorula rubra, R. glutinis, R.marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pullulans.

In an embodiment of the invention, the defensins of the invention may beused as a pharmaceutical compound for treatment of fungal and microbialpathogens in humans and other animals. Diseases and disorders caused byfungal and microbial pathogens include but are not limited to fungalmeningoencephalitis, superficial fungal infections, ringworm, Athlete'sfoot, histoplasmosis, candidiasis, thrush, coccidioidoma, pulmonarycryptococcus, trichosporonosis, piedra, tinea nigra, fungal keratitis,onychomycosis, tinea capitis, chromomycosis, aspergillosis,endobronchial pulmonary aspergillosis, mucormycosis,chromoblastomycosis, dermatophytosis, tinea, fusariosis, pityriasis,mycetoma, pseudallescheriasis, and sporotrichosis.

In particular, the compositions of the invention may be used aspharmaceutical compounds to provide treatment for diseases and disordersassociated with, but not limited to, the following fungal pathogens:Histoplasma capsulatum, Candida spp. (C. albicans, C. tropicalis, C.parapsilosis, C. guilliermondii, C. glabrata/Torulopsis glabrata, C.krusei, C. lusitaniae), Aspergillus fumigatus, A. flavus, A. niger,Rhizopus spp., Rhizomucor spp., Cunninghamella spp., Apophysomyces spp.,Saksenaee spp., Mucor spp., and Absidia spp. Efficacy of thecompositions of the invention as anti-fungal treatments may bedetermined through anti-fungal assays known to one in the art.

The defensins may be administered to a patient through numerous means.Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to about 15 mg/kg (e.g.,0.1 to 20 mg/kg) of active compound is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

“Treatment” is herein defined as the application or administration of atherapeutic agent to a patient, or application or administration of atherapeutic agent to an isolated tissue or cell line from a patient, whohas a disease, a symptom of disease or a predisposition toward adisease, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve or affect the disease, the symptoms ofdisease or the predisposition toward disease. A “therapeutic agent”comprises, but is not limited to, the small molecules, peptides,antibodies, and antisense oligonucleotides of the invention.

The defensins of the invention can be used for any application includingcoating surfaces to target microbes. In this manner, target microbesinclude human pathogens or microorganisms. Surfaces that might be coatedwith the defensins of the invention include carpets and sterile medicalfacilities. Polymer bound polypeptides of the invention may be used tocoat surfaces. Methods for incorporating compositions with antimicrobialproperties into polymers are known in the art. See U.S. Pat. No.5,847,047 herein incorporated by reference.

An isolated polypeptide of the invention can be used as an immunogen togenerate antibodies that bind defensins using standard techniques forpolyclonal and monoclonal antibody preparation. The full-lengthdefensins can be used or, alternatively, the invention providesantigenic peptide fragments of defensins for use as immunogens. Theantigenic peptide of a defensive agent comprises at least 8, preferably10, 15, 20, or amino acid residues of the amino acid sequence shown inSEQ ID NO: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26,27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53,54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 75, 77, 78, 80,81, 83, 84, 86, 87, 89, 90, 92, 93, 95, 96, 98, 99, 101, 102, 104, 105,107, 108, 110, 111, 113, 114, 116, 117, 119, 120, 122, 123, 125, 126,128, 129, 131, 132, 134, 135, 137, 138, 140, 141, 143, 144, 146, 147,149, 150, 152, 153, 155, 156, 158, 159, 161, 162, 164, 165, 167, 168,170, 171, 173, 174, 176, 177, 179, 180, 182, 183, 185, 186, 188, 189,191, 192, 194, 195, 197, 198, 200, 201, 203, 204, 206, 207, 209, 210,212, 213, 215, 216, 218, 219, 221, 222, 224, 225, 227, 228, 230, 231,233, 234, 236, 237, 239, 240, 242, 243, 245, 246, 248, 249, 251, 252,254, 255, 257, 258, 260, 261, 263, 264, 266, 267, 269, 270, 272, 273,275, 276, 278, 279, 281, 282, 284, 285, 287, 288, 290, 291, 293, 294,296, 297, 299, 300, 302, 303, 305, 306, 308, 309, 311, 312, 314, 315,317, 318, 320, 321, 323, 324, 326, 327, 329, 330, 332, 333, 335, 336,338, 339, 341, 342, 344, 345, 347, 348, 350, 351, 353, 354, 356, 357,359, 360, 362, 363, 365, 366, 368, 369, 371, 372, 374, 375, 377, 378,380, 381, 383, 384, 386, 387, 389, 390, 392, 393, 395, 396, 398, 399,401, 402, 404, 405, 407, 408, 410, 411, 413, 414, 416, 417, 419, 420,422, 423, 425, 426, 428, 429, 431, 432, 434, 435, 437, 438, 440, 441,443, 444, 446, 447, 449, 450, 452, 453, 455, 456, 458, 459, 461, 462,464, 466, or 468 and encompasses an epitope of a defensin such that anantibody raised against the peptide forms a specific immune complex withthe antimicrobial polypeptides. Epitopes encompassed by the antigenicpeptide are regions of defensins that are located on the surface of theprotein, e.g., hydrophilic regions, which are readily ascertainable bythose of skill in the art.

Accordingly, another aspect of the invention pertains to anti-defensinpolyclonal and monoclonal antibodies that bind a defensin. Polyclonaldefensin-like antibodies can be prepared by immunizing a suitablesubject (e.g., rabbit, goat, mouse, or other mammal) with an defensiveagent immunogen. The anti-defensin antibody titer in the immunizedsubject can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilizedantimicrobial polypeptides. At an appropriate time after immunization,e.g., when the anti-defensive agent antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al.(1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al.(1985) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld andSell (Alan R. Liss, Inc., New York, N.Y.), pp. 77-96) or triomatechniques. The technology for producing hybridomas is well known (seegenerally Coligan et al., eds. (1994) Current Protocols in Immunology(John Wiley & Sons, Inc., New York, N.Y.); Galfre et al. (1977) Nature266:55052; Kenneth (1980) in Monoclonal Antibodies: A New Dimension InBiological Analyses (Plenum Publishing Corp., NY; and Lerner (1981) YaleJ. Biol. Med., 54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-defensin-like antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with a defensin to thereby isolateimmunoglobulin library members that bind the defensive agent. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening an antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734. The antibodies can be used to identifyhomologs of the defensins of the invention.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Transformation and Regeneration of TransgenicPlants in Maize

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing a defensin nucleotide sequence of the inventionoperably linked to a ubiquitin promoter and the selectable marker genePAT (Wohlleben et al. (1988) Gene 70:25-37), which confers resistance tothe herbicide Bialaphos. Alternatively, the selectable marker gene isprovided on a separate plasmid. Transformation is performed as follows.Media recipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% Clorox bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5-cm target zone in preparation forbombardment.

Preparation of DNA

A plasmid vector comprising a defensin nucleotide sequence of theinvention operably linked to a ubiquitin promoter is made. This plasmidDNA plus plasmid DNA containing a PAT selectable marker is precipitatedonto 1.1 μm (average diameter) tungsten pellets using a CaCl₂precipitation procedure as follows:

100 μl prepared tungsten particles in water

10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)

100 μl 2.5 M CaCl₂

10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for altered defense response defensinactivity, insect resistance, nematode resistance, viral resistance, orfungal resistance.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H₂O); and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511),0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought tovolume with D-I H₂O following adjustment to pH 5.8 with KOH); 3.0 g/lGelrite (added after bringing to volume with D-I H₂O); and 0.85 mg/lsilver nitrate and 3.0 mg/l Bialaphos (both added after sterilizing themedium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycinebrought to volume with polished D-I H₂0) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite (addedafter bringing to volume with D-I H₂O); and 1.0 mg/l indoleacetic acidand 3.0 mg/l Bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinicacid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/lglycine brought to volume with polished D-I H₂O), 0.1 g/1 myo-inositol,and 40.0 g/l sucrose (brought to volume with polished D-I H₂O afteradjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing tovolume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 2 Agrobacterium-Mediated Transformation in Maize

For Agrobacterium-mediated transformation of maize with a defensinnucleotide sequence of the invention operably linked to a ubiquitinpromoter, preferably the method of Zhao is employed (U.S. Pat. No.5,981,840, and PCT patent publication WO98/32326; the contents of whichare hereby incorporated by reference). Briefly, immature embryos areisolated from maize and the embryos contacted with a suspension ofAgrobacterium, where the bacteria are capable of transferring the DNAconstruct containing the defensin nucleotide sequence to at least onecell of at least one of the immature embryos (step 1: the infectionstep). In this step the immature embryos are preferably immersed in anAgrobacterium suspension for the initiation of inoculation. The embryosare co-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). Preferably the immature embryos are cultured onsolid medium following the infection step. Following this co-cultivationperiod an optional “resting” step is contemplated. In this resting step,the embryos are incubated in the presence of at least one antibioticknown to inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step).Preferably the immature embryos are cultured on solid medium withantibiotic, but without a selecting agent, for elimination ofAgrobacterium and for a resting phase for the infected cells. Next,inoculated embryos are cultured on medium containing a selective agentand growing transformed callus is recovered (step 4: the selectionstep). Preferably, the immature embryos are cultured on solid mediumwith a selective agent resulting in the selective growth of transformedcells. The callus is then regenerated into plants (step 5: theregeneration step), and preferably calli grown on selective medium arecultured on solid medium to regenerate the plants.

Example 3 Soybean Embryo Transformation

Soybean embryos are bombarded with a plasmid containing the defensinnucleotide sequences operably linked to a ubiquitin promoter as follows.To induce somatic embryos, cotyledons, 3-5 mm in length dissected fromsurface-sterilized, immature seeds of the soybean cultivar A2872, arecultured in the light or dark at 26° C. on an appropriate agar mediumfor six to ten weeks. Somatic embryos producing secondary embryos arethen excised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos that multiplied as early,globular-staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 ml ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A Du Pont Biolistic PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188), and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising the defensin nucleotidesequence operably linked to the ubiquitin promoter can be isolated as arestriction fragment. This fragment can then be inserted into a uniquerestriction site of the vector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 4 Sunflower Meristem Tissue Transformation

Sunflower meristem tissues are transformed with an expression cassettecontaining the defensin sequence operably linked to a ubiquitin promoteras follows (see also European Patent Number EP 0 486233, hereinincorporated by reference, and Malone-Schoneberg et al. (1994) PlantScience 103:199-207). Mature sunflower seed (Helianthus annuus L.) aredehulled using a single wheat-head thresher. Seeds are surfacesterilized for 30 minutes in a 20% Clorox bleach solution with theaddition of two drops of Tween 20 per 50 ml of solution. The seeds arerinsed twice with sterile distilled water.

Split embryonic axis explants are prepared by a modification ofprocedures described by Schrammeijer et al. (Schrammeijer et al. (1990)Plant Cell Rep. 9: 55-60). Seeds are imbibed in distilled water for 60minutes following the surface sterilization procedure. The cotyledons ofeach seed are then broken off, producing a clean fracture at the planeof the embryonic axis. Following excision of the root tip, the explantsare bisected longitudinally between the primordial leaves. The twohalves are placed, cut surface up, on GBA medium consisting of Murashigeand Skoog mineral elements (Murashige et al. (1962) Physiol. Plant., 15:473-497), Shepard's vitamin additions (Shepard (1980) in EmergentTechniques for the Genetic Improvement of Crops University of MinnesotaPress, St. Paul, Minn.), 40 mg/l adenine sulfate, 30 g/l sucrose, 0.5mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-acetic acid (IAA),0.1 mg/l gibberellic acid (GA3), pH 5.6, and 8 g/l Phytagar.

The explants are subjected to microprojectile bombardment prior toAgrobacterium treatment (Bidney et al. (1992) Plant Mol. Biol. 18:301-313). Thirty to forty explants are placed in a circle at the centerof a 60×20 mm plate for this treatment. Approximately 4.7 mg of 1.8 mmtungsten microprojectiles are resuspended in 25 ml of sterile TE buffer(10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are used perbombardment. Each plate is bombarded twice through a 150 mm nytex screenplaced 2 cm above the samples in a PDS 1000® particle accelerationdevice.

Disarmed Agrobacterium tumefaciens strain EHA105 is used in alltransformation experiments. A binary plasmid vector comprising theexpression cassette that contains the defensin gene operably linked tothe ubiquitin promoter is introduced into Agrobacterium strain EHA105via freeze-thawing as described by Holsters et al. (1978) Mol. Gen.Genet. 163:181-187. This plasmid further comprises a kanamycinselectable marker gene (i.e., nptII). Bacteria for plant transformationexperiments are grown overnight (28° C. and 100 RPM continuousagitation) in liquid YEP medium (10 gm/l yeast extract, 10 gm/lBactopeptone, and 5 gm/l NaCl, pH 7.0) with the appropriate antibioticsrequired for bacterial strain and binary plasmid maintenance. Thesuspension is used when it reaches an OD₆₀₀ of about 0.4 to 0.8. TheAgrobacterium cells are pelleted and resuspended at a final OD₆₀₀ of 0.5in an inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm/l NH₄Cl,and 0.3 gm/l MgSO₄.

Freshly bombarded explants are placed in an Agrobacterium suspension,mixed, and left undisturbed for 30 minutes. The explants are thentransferred to GBA medium and co-cultivated, cut surface down, at 26° C.and 18-hour days. After three days of co-cultivation, the explants aretransferred to 374B (GBA medium lacking growth regulators and a reducedsucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/lkanamycin sulfate. The explants are cultured for two to five weeks onselection and then transferred to fresh 374B medium lacking kanamycinfor one to two weeks of continued development. Explants withdifferentiating, antibiotic-resistant areas of growth that have notproduced shoots suitable for excision are transferred to GBA mediumcontaining 250 mg/l cefotaxime for a second 3-day phytohormonetreatment. Leaf samples from green, kanamycin-resistant shoots areassayed for the presence of NPTII by ELISA and for the presence oftransgene expression by assaying for defensin-like activity.

NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in vitro-grownsunflower seedling rootstock. Surface sterilized seeds are germinated in48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3%gelrite, pH 5.6) and grown under conditions described for explantculture. The upper portion of the seedling is removed, a 1 cm verticalslice is made in the hypocotyl, and the transformed shoot inserted intothe cut. The entire area is wrapped with parafilm to secure the shoot.Grafted plants can be transferred to soil following one week of in vitroculture. Grafts in soil are maintained under high humidity conditionsfollowed by a slow acclimatization to the greenhouse environment.Transformed sectors of T₀ plants (parental generation) maturing in thegreenhouse are identified by NPTII ELISA and/or by defensin-likeactivity analysis of leaf extracts while transgenic seeds harvested fromNPTII-positive T₀ plants are identified by defensin-like activityanalysis of small portions of dry seed cotyledon.

An alternative sunflower transformation protocol allows the recovery oftransgenic progeny without the use of chemical selection pressure. Seedsare dehulled and surface-sterilized for 20 minutes in a 20% Cloroxbleach solution with the addition of two to three drops of Tween 20 per100 ml of solution, then rinsed three times with distilled water.Sterilized seeds are imbibed in the dark at 26° C. for 20 hours onfilter paper moistened with water. The cotyledons and root radical areremoved, and the meristem explants are cultured on 374E (GBA mediumconsisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate, 3%sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA, and 0.8% Phytagarat pH 5.6) for 24 hours under the dark. The primary leaves are removedto expose the apical meristem, around 40 explants are placed with theapical dome facing upward in a 2 cm circle in the center of 374M (GBAmedium with 1.2% Phytagar), and then cultured on the medium for 24 hoursin the dark.

Approximately 18.8 mg of 1.8 μm tungsten particles are resuspended in150 μl absolute ethanol. After sonication, 8 μl of it are dropped on thecenter of the surface of macrocarrier. Each plate is bombarded twicewith 650 PSI rupture discs in the first shelf at 26 mm of Hg helium gunvacuum.

The plasmid of interest is introduced into Agrobacterium tumefaciensstrain EHA105 via freeze thawing as described previously. The pellet ofovernight-grown bacteria at 28° C. in a liquid YEP medium (10 g/l yeastextract, 10 g/l Bactopeptone, and 5 g/l NaCl, pH 7.0) in the presence of50 μg/l kanamycin is resuspended in an inoculation medium (12.5 mM 2-mM2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH₄Cl and 0.3 g/l MgSO₄at pH 5.7) to reach a final concentration of 4.0 at OD 600.Particle-bombarded explants are transferred to GBA medium (374E), and adroplet of bacteria suspension is placed directly onto the top of themeristem. The explants are co-cultivated on the medium for 4 days, afterwhich the explants are transferred to 374C medium (GBA with 1% sucroseand no BAP, IAA, GA3 and supplemented with 250 μg/ml cefotaxime). Theplantlets are cultured on the medium for about two weeks under 16-hourday and 26° C. incubation conditions.

Explants (around 2 cm long) from two weeks of culture in 374C medium arescreened for defensin-like activity using assays known in the art. Afterpositive (i.e., for defensin expression) explants are identified, thoseshoots that fail to exhibit defensin-like activity are discarded, andevery positive explant is subdivided into nodal explants. One nodalexplant contains at least one potential node. The nodal segments arecultured on GBA medium for three to four days to promote the formationof auxiliary buds from each node. Then they are transferred to 374Cmedium and allowed to develop for an additional four weeks. Developingbuds are separated and cultured for an additional four weeks on 374Cmedium. Pooled leaf samples from each newly recovered shoot are screenedagain by the appropriate defensin-like protein activity assay. At thistime, the positive shoots recovered from a single node will generallyhave been enriched in the transgenic sector detected in the initialassay prior to nodal culture.

Recovered shoots positive for defensin-like activity expression aregrafted to Pioneer hybrid 6440 in vitro-grown sunflower seedlingrootstock. The rootstocks are prepared in the following manner. Seedsare dehulled and surface-sterilized for 20 minutes in a 20% Cloroxbleach solution with the addition of two to three drops of Tween 20 per100 ml of solution, and are rinsed three times with distilled water. Thesterilized seeds are germinated on the filter moistened with water forthree days, then they are transferred into 48 medium (half-strength MSsalt, 0.5% sucrose, 0.3% gelrite pH 5.0) and grown at 26° C. under thedark for three days, then incubated at 16-hour-day culture conditions.The upper portion of selected seedling is removed, a vertical slice ismade in each hypocotyl, and a transformed shoot is inserted into aV-cut. The cut area is wrapped with parafilm. After one week of cultureon the medium, grafted plants are transferred to soil. In the first twoweeks, they are maintained under high humidity conditions to acclimatizeto a greenhouse environment.

Example 5 Assaying Defensin-Like Activity

The polypeptides described herein may be produced using any number ofmethods known to those skilled in the art. Such methods include, but arenot limited to, expression in bacteria, eukaryotic cell cultures, inplanta, and viral expression systems in suitably infected organisms orcell lines. The instant polypeptides may be expressed either asfull-length polypeptides, mature forms, or as fusion proteins bycovalent attachment to a variety of enzymes, proteins, or affinity tags.Common fusion protein partners include, but are not limited to,glutathione-S-transferase, thioredoxin, maltose binding protein,hexahistidine polypeptides, and chitin binding protein. The fusionproteins may be engineered with a protease recognition site at thefusion point so that fusion partners can be separated by proteasedigestion to yield intact mature peptides. Examples of such proteasesinclude, but are not limited to, thrombin, enterokinase, and factor Xa.Indeed, any protease which specifically cleaves the peptide connectingthe fusion protein and polypeptide of the invention can be used.

Purification of the polypeptides of the invention may utilize any numberof separation technologies known to those skilled in the art of proteinpurification. Examples of such methods include, but are not limited to,homogenization, filtration, centrifugation, heat denaturation, ammoniumsulfate precipitation, desalting, pH precipitation, ion exchangechromatography, hydrophobic interaction chromatography, and affinitychromatography. When the polypeptides of the invention are expressed asfusion proteins, the purification protocol may include the use of anaffinity resin specific for the fusion protein partner or for thepolypeptide of interest. Additional suitable affinity resins may besynthesized by linking the appropriate ligands to a suitable resin suchas Sepharose-4B.

Crude, partially purified, or purified polypeptides of the invention,either alone or as a fusion protein, may be utilized in assays to verifyexpression levels of functional plant defensins in host cells andtransgenic plants. Assays may be conducted under well known experimentalconditions which permit optimal enzymatic activity. See, for example,assays for plant defensin activities presented by Thevissen, K et al.(1996) J. Biol. Chem. 271:15018-15025 and WO 00/68405, hereinincorporated by reference.

Example 6 Bioassay Testing the Pesticidal Activity of PolypeptidesAgainst Southern Corn Rootworm (SCRW) and Western Corn Rootworm (WCRW)

Bio-Serv diet (catalog number F9800B, from: BIOSERV, EntomologyDivision, One 8^(th) Street, Suite 1, Frenchtown, N.J. 08825) isdispensed in 128-well CD International Bioassay trays (catalog numberBIO-BA-128 from CD International, Pitman, N.J. 08071).

Protein samples are applied topically to the diet surface. Enough samplematerial is supplied to provide for replicate observations per sample.The trays are allowed to dry. Rootworms are dispensed into the wells ofthe bioassay trays. A lid (catalog number BIO-CV-16, CD International,Pitman, N.J., 08071) is placed on each tray, and the trays are placed inan incubator at 26° C. for 4 to 7 days.

For the evaluation of pesticidal activity against SCRW and WCRW, insectsare exposed to a solution comprising either buffer (50 mM carbonatebuffer (pH 10)) or a solution of protein sample at selected doses, forexample, 50 or 5.0 μg/cm².

The bioassays are then scored by counting “live” versus “dead” larvae.Mortality is calculated as a percentage of dead larvae out of the totalnumber of larvae tested.

Example 7 Bioassay Testing Pesticidal Activity of Polypeptides againstthe Colorado Potato Beetle (Leptinotarsa decemlineata)

Briefly, bioassay parameters are as follows: Bio-Serv diet (catalognumber F9800B, from: BIOSERV, Entomology Division, One 8th Street, Suite1, Frenchtown, N.J. 08825) is dispensed in a 96 well microtiter plate(catalog number 353918, Becton Dickinson, Franklin Lakes, N.J.07417-1886) having a surface area of 0.33 cm². Protein samples of theinvention are applied topically to the diet surface. Enough samplematerial is supplied to provide for 8 observations/sample. After thesamples dry, 1 Colorado potato beetle neonate is added to each wellproviding for a total of 8 larvae/sample. A Mylar® lid (Clear LamPackaging, Inc., 1950 Pratt Blvd., Elk Grove Village, Ill. 60007-5993)is affixed to each tray. Bioassay trays are placed in an incubator at25° C. The test is scored for mortality on the 7th day following liveinfesting.

Example 8 Bioassay Testing Pesticidal Activity of Polypeptides againstLepidopterans

Neonate larvae are reared according to standard protocols, such as thosepublished by Czapla and Lang, J. Economic Entomology 83:2480-2485(1990). Test compounds are either applied topically to the diet orincorporated into the larvae diet (see Czapla and Lang, J. EconomicEntomology 83:2480-2485 (1990)). The larvae diet is dispensed tobioassay trays. One larva is applied per well of the bioassay tray.Weight and mortality are recorded 7 days following the start of thetest.

Example 9 Homopteran Membrane Feeding Bioassay for Screening Proteins

This assay can be used for a variety of homopterans. The assay involvestrapping the sample protein between two layers of maximally stretchedparafilm which act as a sachet on top of a small vessel containing theinsect of choice.

The assay is prepared as follows: 1 cm diameter polystyrene tubing iscut into 15 mm lengths. One end of the tube is then capped with a finemesh screen. Five insects are then added to the chamber after which thefirst layer of parafilm is stretched over the remaining open end. 25 μlof sample (polypeptide in a 5% sucrose solution containing McCormickgreen food coloring) is then placed on top of the stretched parafilm. Asecond layer of parafilm is then stretched by hand and placed over thesample. The sample is spread between the two layers of parafilm to makea continuous sachet on which the insects feed. The sachet is thencovered tightly with saran wrap to prevent evaporation and produce aslightly pressurized sample. The assay tubes are monitored for insectreproduction and death on a 24 hour basis and compared to the 5% sucrosecontrol.

Example 10 SCN Bioassay of Transgenic T0 Events

Soybean Cyst Nematodes (SCN) are used to infest transgenic T0 soybeanplants in soil. SCN egg inoculum is acquired by harvesting cysts fromplants infested 4-6 weeks earlier. Briefly, the soil is rinsed from theroots and passed through nested 20 mesh and 60 mesh screens. Thematerial retained by the 20 mesh screen is discarded but the materialretained by the 60 mesh screen is washed thoroughly and the creamy whitecysts are recovered (older brown cysts are ignored). Similarly, theplant's root system is scrubbed against the 20 mesh screen nested overthe 60 mesh screen. Cysts are harvested from the debris on the 60 meshscreen. Eggs are released from the cysts by means of a douncehomogenizer in the presence of 0.5% Clorox for 2.5 minutes. Followingthis treatment the eggs are washed with sterile water from thehomogenizer onto the surface of a 200 mesh screen. The eggs are thenrinsed in water for an additional 5 minutes. Eggs are transferred to a50 ml conical tube and counted. The eggs are diluted to 5000 eggs/ml.Plants grown in 15 cm conical tubes are inoculated with about 5000 eggs.Plants are maintained in a 26° C. growth chamber with 12:12 light:darkcycle for 1 month prior to harvest and counting of cysts.

Example 11 Bioactivity of Polypeptides Against Fungal Pathogens

The proteins of the invention are suspended in dH2O to a finalconcentration of about 4 μg/μl. 12 μg of purified protein is added to200 μl of ½ strength potato dextrose broth (PDB) containing a sporesuspension of the fungal pathogen to be tested. The spore suspensioncontains approximately 2500 spores/ml. This results in a stock solutionwith a starting concentration of 10 μM. A 0.5× dilution series for theprotein sample to be tested from 10 μM through to about 0.05 μM isprepared by removing 100 μl of the 10 μM stock and adding it to 100 μlof spore suspension (2500 spores/ml), mixing thoroughly to achieve a 5μM protein sample concentration, transferring 100 μl of the 5 μMsuspension to a fresh 100 μl spore suspension etc., until about 0.005 μMis reached. Two replicates per pathogen are performed. The fungal assayplate is scored for inhibition of fungal growth after a 48 hourincubation at 28° C. Inhibition of fungal growth is defined as little tono spore germination without detectable hyphae growth.

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example, for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated nucleic acid molecule comprising a nucleotide sequencethat encodes a polypeptide that has defensin-like activity, wherein saidpolypeptide comprises an amino acid sequence that has at least 95percent sequence identity to the amino acid sequence set forth in SEQ IDNO:81.
 2. A DNA construct comprising the nucleic acid molecule of claim1, wherein said nucleotide sequence is operably linked to a promoterthat drives expression in a host cell.
 3. A transformed host cellcomprising in its genome at least one stably incorporated DNA constructof claim
 2. 4. The host cell of claim 3, wherein said host cell is aplant cell.
 5. A plant comprising the host cell of claim
 3. 6.Transformed seed of the plant of claim 5, wherein said seed comprisessaid DNA construct.
 7. The isolated nucleic acid molecule of claim 1,wherein said nucleotide sequence is set forth in SEQ ID NO:79 betweennucleotides 176 and
 322. 8. The isolated nucleic acid molecule of claim7, wherein said nucleotide sequence is set forth in SEQ ID NO:79.
 9. Theisolated nucleic acid molecule of claim 7, wherein said nucleotidesequence encodes a polypeptide comprising the amino acid sequence setforth in SEQ ID NO:81.
 10. The plant of claim 5, wherein said plant hasenhanced resistance to a disease caused by a fungal pathogen.
 11. Theplant of claim 10, wherein said fungal pathogen is selected from thegroup consisting of Colletotrichum graminicola, Diplodia maydis,Fusarium graminearum, and Fusarium moniliforme.
 12. The plant of claim10, wherein said plant is selected from the group consisting of soybean,canola, alfalfa, wheat, sunflower, corn, and sorghum.
 13. The plant ofclaim 5, wherein said plant has enhanced resistance to a disease causedby a nematode pathogen.
 14. A method for enhancing disease resistance ina plant, said method comprising transforming said plant with at leastone nucleotide construct comprising a nucleotide sequence encoding apolypeptide having at least 95% sequence identity to the sequence setforth in SEQ ID NO: 81 operably linked to a promoter that drivesexpression in a plant, and regenerating said transformed plant.
 15. Themethod of claim 14, wherein said disease resistance is resistance to adisease caused by a fungal pathogen.
 16. The method of claim 15, whereinsaid fungal pathogen is selected from the group consisting ofColletotrichum graminicola, Diplodia maydis, Fusarium graminearum, andFusarium moniliforme.
 17. The method of claim 14, wherein said diseaseresistance is resistance to a disease caused by a nematode pathogen. 18.The method of claim 14, wherein said nucleotide sequence is the sequenceset forth in SEQ ID NO:79.
 19. The method of claim 14, wherein saidplant is selected from the group consisting of soybean, canola, alfalfa,wheat, sunflower, corn, and sorghum.